Compounds and compositions for intracellular delivery of therapeutic agents

ABSTRACT

The disclosure features novel lipids and compositions involving the same. Nanoparticle compositions include a novel lipid as well as additional lipids such as phospholipids, structural lipids, and PEG lipids. Nanoparticle compositions further including therapeutic and/or prophylactics such as RNA are useful in the delivery of therapeutic and/or prophylactics to mammalian cells or organs to, for example, regulate polypeptide, protein, or gene expression.

RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 15/476,253 filed onMar. 31, 2017, which is a continuation application of InternationalApplication PCT/US2016/052352, having an international filing date ofSep. 16, 2016, which claims priority to, and the benefit of, U.S.Provisional Application Nos. 62/220,085, filed Sep. 17, 2015;62/220,091, filed Sep. 17, 2015; 62/252,316, filed Nov. 6, 2015;62/253,433, filed Nov. 10, 2015; 62/266,460, filed Dec. 11, 2015;62/333,557, filed May 9, 2016; 62/382,740, filed Sep. 1, 2016; and62/393,940, filed Sep. 13, 2016; the entire contents of each of whichare incorporated herein by reference in their entireties.

INCORPORATION-BY-REFERENCE OF SEQUENCE LISTING

The contents of the text file named “MRNA014001WOST25.txt”, which wascreated on Dec. 2, 2016 and is 1 KB in size, are hereby incorporated byreference in their entireties.

FIELD OF DISCLOSURE

The present disclosure provides novel compounds, compositions comprisingsuch compounds, and methods involving lipid nanoparticle compositions todeliver one or more therapeutic and/or prophylactics to and/or producepolypeptides in mammalian cells or organs. In addition to a novel lipid,lipid nanoparticle compositions of the disclosure may include one ormore cationic and/or ionizable amino lipids, phospholipids includingpolyunsaturated lipids, PEG lipids, structural lipids, and/ortherapeutic and/or prophylactics in specific fractions.

BACKGROUND OF THE DISCLOSURE

The effective targeted delivery of biologically active substances suchas small molecule drugs, proteins, and nucleic acids represents acontinuing medical challenge. In particular, the delivery of nucleicacids to cells is made difficult by the relative instability and lowcell permeability of such species. Thus, there exists a need to developmethods and compositions to facilitate the delivery of therapeuticand/or prophylactics such as nucleic acids to cells.

Lipid-containing nanoparticle compositions, liposomes, and lipoplexeshave proven effective as transport vehicles into cells and/orintracellular compartments for biologically active substances such assmall molecule drugs, proteins, and nucleic acids. Such compositionsgenerally include one or more “cationic” and/or amino (ionizable)lipids, phospholipids including polyunsaturated lipids, structurallipids (e.g., sterols), and/or lipids containing polyethylene glycol(PEG lipids). Cationic and/or ionizable lipids include, for example,amine-containing lipids that can be readily protonated. Though a varietyof such lipid-containing nanoparticle compositions have beendemonstrated, improvements in safety, efficacy, and specificity arestill lacking.

SUMMARY OF THE DISCLOSURE

The present disclosure provides novel compounds and compositions andmethods involving the same.

A first aspect of the disclosure relates to compounds of Formula (I):

or a salt or isomer thereof, wherein:

R₁ is selected from the group consisting of C₅₋₃₀ alkyl, C₅₋₂₀ alkenyl,—R*YR″, —YR″, and —R″M′R′;

-   -   R₂ and R₃ are independently selected from the group consisting        of H, C₁₋₁₄ alkyl, C₂₋₁₄ alkenyl, —R*YR″, —YR″, and —R*OR″, or        R₂ and R₃, together with the atom to which they are attached,        form a heterocycle or carbocycle;    -   R₄ is selected from the group consisting of a C₃₋₆ carbocycle,        —(CH₂)_(n)Q, —(CH₂)_(n)CHQR, —CHQR, —CQ(R)₂, and unsubstituted        C₁₋₆ alkyl, where Q is selected from a carbocycle, heterocycle,        —OR, —O(CH₂)_(n)N(R)₂, —C(O)OR, —OC(O)R, —CX₃, —CX₂H, —CXH₂,        —CN, —N(R)₂, —C(O)N(R)₂, —N(R)C(O)R, —N(R)S(O)₂R,        —N(R)C(O)N(R)₂, —N(R)C(S)N(R)₂, —N(R)R₈, —O(CH₂)_(n)OR,        —N(R)C(═NR₉)N(R)₂, —N(R)C(═CHR₉)N(R)₂, —OC(O)N(R)₂, —N(R)C(O)OR,        —N(OR)C(O)R, —N(OR)S(O)₂R, —N(OR)C(O)OR, —N(OR)C(O)N(R)₂,        —N(OR)C(S)N(R)₂, —N(OR)C(═NR₉)N(R)₂, —N(OR)C(═CHR₉)N(R)₂,        —C(═NR₉)N(R)₂, —C(═NR₉)R, —C(O)N(R)OR, and —C(R)N(R)₂C(O)OR, and        each n is independently selected from 1, 2, 3, 4, and 5;    -   each R₅ is independently selected from the group consisting of        C₁₋₃ alkyl, C₂₋₃ alkenyl, and H;    -   each R₆ is independently selected from the group consisting of        C₁₋₃ alkyl, C₂₋₃ alkenyl, and H;    -   M and M′ are independently selected from —C(O)O—, —OC(O)—,        —C(O)N(R′)—, —N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—,        —CH(OH)—, —P(O)(OR′)O—, —S(O)₂—, —S—S—, an aryl group, and a        heteroaryl group;    -   R₇ is selected from the group consisting of C₁₋₃ alkyl, C₂₋₃        alkenyl, and H;    -   R₈ is selected from the group consisting of C₃₋₆ carbocycle and        heterocycle;    -   R₉ is selected from the group consisting of H, CN, NO₂, C₁₋₆        alkyl, —OR, —S(O)₂R, —S(O)₂N(R)₂, C₂₋₆ alkenyl, C₃₋₆ carbocycle        and heterocycle;    -   each R is independently selected from the group consisting of        C₁₋₃ alkyl, C₂₋₃ alkenyl, and H;    -   each R′ is independently selected from the group consisting of        C₁₋₁₈ alkyl, C₂₋₁₈ alkenyl, —R*YR″, —YR″, and H;    -   each R″ is independently selected from the group consisting of        C₃₋₁₄ alkyl and C₃₋₁₄ alkenyl;    -   each R* is independently selected from the group consisting of        C₁₋₁₂ alkyl and C₂₋₁₂ alkenyl;    -   each Y is independently a C₃₋₆ carbocycle;    -   each X is independently selected from the group consisting of F,        Cl, Br, and I; and    -   m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13.

In some embodiments, a subset of compounds of Formula (I) includes thosein which when R₄ is —(CH₂)_(n)Q, —(CH₂)_(n)CHQR, —CHQR, or —CQ(R)₂, then(i) Q is not —N(R)₂ when n is 1, 2, 3, 4 or 5, or (ii) Q is not 5, 6, or7-membered heterocycloalkyl when n is 1 or 2.

In another embodiments, another subset of compounds of Formula (I)includes those in which

-   -   R₁ is selected from the group consisting of C₅₋₃₀ alkyl, C₅₋₂₀        alkenyl, —R*YR″, —YR″, and —R″M′R′;    -   R₂ and R₃ are independently selected from the group consisting        of H, C₁₋₁₄ alkyl, C₂₋₁₄ alkenyl, —R*YR″, —YR″, and —R*OR″, or        R₂ and R₃, together with the atom to which they are attached,        form a heterocycle or carbocycle;    -   R₄ is selected from the group consisting of a C₃₋₆ carbocycle,        —(CH₂)_(n)Q, —(CH₂)_(n)CHQR, —CHQR, —CQ(R)₂, and unsubstituted        C₁₋₆ alkyl, where Q is selected from a C₃₋₆ carbocycle, a 5- to        14-membered heteroaryl having one or more heteroatoms selected        from N, O, and S, —OR, —O(CH₂)_(n)N(R)₂, —C(O)OR, —OC(O)R, —CX₃,        —CX₂H, —CXH₂, —CN, —C(O)N(R)₂, —N(R)C(O)R, —N(R)S(O)₂R,        —N(R)C(O)N(R)₂, —N(R)C(S)N(R)₂, —CRN(R)₂C(O)OR, —N(R)R₈,        —O(CH₂)_(n)OR, —N(R)C(═NR₉)N(R)₂, —N(R)C(═CHR₉)N(R)₂,        —OC(O)N(R)₂, —N(R)C(O)OR, —N(OR)C(O)R, —N(OR)S(O)₂R,        —N(OR)C(O)OR, —N(OR)C(O)N(R)₂, —N(OR)C(S)N(R)₂,        —N(OR)C(═NR₉)N(R)₂, —N(OR)C(═CHR₉)N(R)₂, —C(═NR₉)N(R)₂,        —C(═NR₉)R, —C(O)N(R)OR, and a 5- to 14-membered heterocycloalkyl        having one or more heteroatoms selected from N, O, and S which        is substituted with one or more substituents selected from oxo        (═O), OH, amino, mono- or di-alkylamino, and C₁₋₃ alkyl, and        each n is independently selected from 1, 2, 3, 4, and 5;

each R₅ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R₆ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

M and M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—,—N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—,—S(O)₂—, —S—S—, an aryl group, and a heteroaryl group;

R₇ is selected from the group consisting of C₁-3 alkyl, C₂-3 alkenyl,and H;

R₈ is selected from the group consisting of C₃-6 carbocycle andheterocycle;

R₉ is selected from the group consisting of H, CN, NO₂, C₁₋₆ alkyl, —OR,—S(O)₂R, —S(O)₂N(R)₂, C₂₋₆ alkenyl, C₃₋₆ carbocycle and heterocycle;

each R is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R′ is independently selected from the group consisting of C₁₋₈alkyl, C₂₋₁₈ alkenyl, —R*YR″, —YR″, and H;

each R″ is independently selected from the group consisting of C₃₋₁₄alkyl and C₃₋₁₄ alkenyl;

each R* is independently selected from the group consisting of C₁₋₁₂alkyl and C₂₋₁₂ alkenyl;

each Y is independently a C₃₋₆ carbocycle;

each X is independently selected from the group consisting of F, Cl, Br,and I; and

m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13,

or salts or isomers thereof.

In yet another embodiments, another subset of compounds of Formula (I)includes those in which

R₁ is selected from the group consisting of C₅₋₃₀ alkyl, C₅₋₂₀ alkenyl,—R*YR″, —YR″, and —R″M′R′;

R₂ and R₃ are independently selected from the group consisting of H,C₁₋₁₄ alkyl, C₂₋₁₄ alkenyl, —R*YR″, —YR″, and —R*OR″, or R₂ and R₃,together with the atom to which they are attached, form a heterocycle orcarbocycle;

R₄ is selected from the group consisting of a C₃₋₆ carbocycle,—(CH₂)_(n)Q, —(CH₂)_(n)CHQR, —CHQR, —CQ(R)₂, and unsubstituted C₁₋₆alkyl, where Q is selected from a C₃₋₆ carbocycle, a 5- to 14-memberedheterocycle having one or more heteroatoms selected from N, O, and S,—OR, —O(CH₂)_(n)N(R)₂, —C(O)OR, —OC(O)R, —CX₃, —CX₂H, —CXH₂, —CN,—C(O)N(R)₂, —N(R)C(O)R, —N(R)S(O)₂R, —N(R)C(O)N(R)₂, —N(R)C(S)N(R)₂,—CRN(R)₂C(O)OR, —N(R)R₈, —O(CH₂)_(n)OR, —N(R)C(═NR₉)N(R)₂,—N(R)C(═CHR₉)N(R)₂, —OC(O)N(R)₂, —N(R)C(O)OR, —N(OR)C(O)R, —N(OR)S(O)₂R,—N(OR)C(O)OR, —N(OR)C(O)N(R)₂, —N(OR)C(S)N(R)₂, —N(OR)C(═NR₉)N(R)₂,—N(OR)C(═CHR₉)N(R)₂, —C(═NR₉)R, —C(O)N(R)OR, and —C(═NR₉)N(R)₂, and eachn is independently selected from 1, 2, 3, 4, and 5; and when Q is a 5-to 14-membered heterocycle and (i) R₄ is —(CH₂)_(n)Q in which n is 1 or2, or (ii) R₄ is —(CH₂)_(n)CHQR in which n is 1, or (iii) R₄ is —CHQR,and —CQ(R)₂, then Q is either a 5- to 14-membered heteroaryl or 8- to14-membered heterocycloalkyl;

each R₅ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R₆ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

M and M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—,—N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—,—S(O)₂—, —S—S—, an aryl group, and a heteroaryl group;

R₇ is selected from the group consisting of C₁₋₃ alkyl, C₂₋₃ alkenyl,and H;

R₈ is selected from the group consisting of C₃₋₆ carbocycle andheterocycle;

R₉ is selected from the group consisting of H, CN, NO₂, C₁₋₆ alkyl, —OR,—S(O)₂R, —S(O)₂N(R)₂, C₂₋₆ alkenyl, C₃₋₆ carbocycle and heterocycle;

each R is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R′ is independently selected from the group consisting of C₁₋₈alkyl, C₂₋₁₈ alkenyl, —R*YR″, —YR″, and H;

each R″ is independently selected from the group consisting of C₃₋₁₄alkyl and C₃₋₁₄ alkenyl;

each R* is independently selected from the group consisting of C₁₋₁₂alkyl and C₂₋₁₂ alkenyl;

each Y is independently a C₃₋₆ carbocycle;

each X is independently selected from the group consisting of F, Cl, Br,and I; and

m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13,

or salts or isomers thereof.

In still another embodiments, another subset of compounds of Formula (I)includes those in which

R₁ is selected from the group consisting of C₅₋₃₀ alkyl, C₅₋₂₀ alkenyl,—R*YR″, —YR″, and —R″M′R′;

R₂ and R₃ are independently selected from the group consisting of H,C₁₋₁₄ alkyl, C₂₋₁₄ alkenyl, —R*YR″, —YR″, and —R*OR″, or R₂ and R₃,together with the atom to which they are attached, form a heterocycle orcarbocycle;

R₄ is selected from the group consisting of a C₃₋₆ carbocycle,—(CH₂)_(n)Q, —(CH₂)_(n)CHQR, —CHQR, —CQ(R)₂, and unsubstituted C₁₋₆alkyl, where Q is selected from a C₃₋₆ carbocycle, a 5- to 14-memberedheteroaryl having one or more heteroatoms selected from N, O, and S,—OR, —O(CH₂)_(n)N(R)₂, —C(O)OR, —OC(O)R, —CX₃, —CX₂H, —CXH₂, —CN,—C(O)N(R)₂, —N(R)C(O)R, —N(R)S(O)₂R, —N(R)C(O)N(R)₂, —N(R)C(S)N(R)₂,—CRN(R)₂C(O)OR, —N(R)R₈, —O(CH₂)_(n)OR, —N(R)C(═NR₉)N(R)₂,—N(R)C(═CHR₉)N(R)₂, —OC(O)N(R)₂, —N(R)C(O)OR, —N(OR)C(O)R, —N(OR)S(O)₂R,—N(OR)C(O)OR, —N(OR)C(O)N(R)₂, —N(OR)C(S)N(R)₂, —N(OR)C(═NR₉)N(R)₂,—N(OR)C(═CHR₉)N(R)₂, —C(═NR₉)R, —C(O)N(R)OR, and —C(═NR₉)N(R)₂, and eachn is independently selected from 1, 2, 3, 4, and 5;

each R₅ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R₆ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

M and M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—,—N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—,—S(O)₂—, —S—S—, an aryl group, and a heteroaryl group;

R₇ is selected from the group consisting of C₁₋₃ alkyl, C₂₋₃ alkenyl,and H;

R₈ is selected from the group consisting of C₃₋₆ carbocycle andheterocycle;

R₉ is selected from the group consisting of H, CN, NO₂, C₁₋₆ alkyl, —OR,—S(O)₂R, —S(O)₂N(R)₂, C₂₋₆ alkenyl, C₃₋₆ carbocycle and heterocycle;

each R is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R′ is independently selected from the group consisting of C₁₋₁₈alkyl, C₂₋₁₈ alkenyl, —R*YR″, —YR″, and H;

each R″ is independently selected from the group consisting of C₃₋₁₄alkyl and C₃₋₁₄ alkenyl;

each R* is independently selected from the group consisting of C₁₋₁₂alkyl and C₂₋₁₂ alkenyl;

each Y is independently a C₃₋₆ carbocycle;

each X is independently selected from the group consisting of F, Cl, Br,and I; and

m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13,

or salts or isomers thereof.

In yet another embodiments, another subset of compounds of Formula (I)includes those in which

R₁ is selected from the group consisting of C₅₋₃₀ alkyl, C₅₋₂₀ alkenyl,—R*YR″, —YR″, and —R″M′R′;

R₂ and R₃ are independently selected from the group consisting of H,C₂₋₁₄ alkyl, C₂₋₁₄ alkenyl, —R*YR″, —YR″, and —R*OR″, or R₂ and R₃,together with the atom to which they are attached, form a heterocycle orcarbocycle;

R₄ is —(CH₂)_(n)Q or —(CH₂)_(n)CHQR, where Q is —N(R)₂, and n isselected from 3, 4, and 5;

each R₅ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R₆ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

M and M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—,—N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—,—S(O)₂—, —S—S—, an aryl group, and a heteroaryl group;

R₇ is selected from the group consisting of C₁₋₃ alkyl, C₂₋₃ alkenyl,and H;

each R is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R′ is independently selected from the group consisting of C₁₋₈alkyl, C₂₋₁₈ alkenyl, —R*YR″, —YR″, and H;

each R″ is independently selected from the group consisting of C₃₋₁₄alkyl and C₃₋₁₄ alkenyl;

each R* is independently selected from the group consisting of C₁₋₁₂alkyl and C₁₋₁₂ alkenyl;

each Y is independently a C₃₋₆ carbocycle;

each X is independently selected from the group consisting of F, Cl, Br,and I; and

m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13,

or salts or isomers thereof.

In still another embodiments, another subset of compounds of Formula (I)includes those in which

R₁ is selected from the group consisting of C₅₋₃₀ alkyl, C₅₋₂₀ alkenyl,—R*YR″, —YR″, and —R″M′R′;

R₂ and R₃ are independently selected from the group consisting of C₁₋₁₄alkyl, C₂₋₁₄ alkenyl, —R*YR″, —YR″, and —R*OR″, or R₂ and R₃, togetherwith the atom to which they are attached, form a heterocycle orcarbocycle;

R₄ is selected from the group consisting of —(CH₂)_(n)Q, —(CH₂)_(n)CHQR,—CHQR, and —CQ(R)₂, where Q is —N(R)₂, and n is selected from 1, 2, 3,4, and 5;

each R₅ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R₆ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

M and M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—,—N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—,—S(O)₂—, —S—S—, an aryl group, and a heteroaryl group;

R₇ is selected from the group consisting of C₁₋₃ alkyl, C₂₋₃ alkenyl,and H;

each R is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R′ is independently selected from the group consisting of C₁₋₁₈alkyl, C₂₋₁₈ alkenyl, —R*YR″, —YR″, and H;

each R″ is independently selected from the group consisting of C₃₋₁₄alkyl and C₃₋₁₄ alkenyl;

each R* is independently selected from the group consisting of C₁₋₁₂alkyl and C₁₋₁₂ alkenyl;

each Y is independently a C₃₋₆ carbocycle;

each X is independently selected from the group consisting of F, Cl, Br,and I; and

m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13,

or salts or isomers thereof.

In certain embodiments, a subset of compounds of Formula (I) includesthose of Formula (IA):

or a salt or isomer thereof, wherein 1 is selected from 1, 2, 3, 4, and5; m is selected from 5, 6, 7, 8, and 9; M₁ is a bond or M′; R₄ isunsubstituted C₁₋₃ alkyl, or —(CH₂)_(n)Q, in which Q is OH,—NHC(S)N(R)₂, —NHC(O)N(R)₂, —N(R)C(O)R, —N(R)S(O)₂R, —N(R)R₈,—NHC(═NR₉)N(R)₂, —NHC(═CHR₉)N(R)₂, —OC(O)N(R)₂, —N(R)C(O)OR, heteroarylor heterocycloalkyl; M and M′ are independently selectedfrom —C(O)O—, —OC(O)—, —C(O)N(R′)—, —P(O)(OR′)O—, —S—S—, an aryl group,and a heteroaryl group; and R₂ and R₃ are independently selected fromthe group consisting of H, C₁-14 alkyl, and C₂₋₁₄ alkenyl.

In certain embodiments, a subset of compounds of Formula (I) includesthose of Formula (II):

or a salt or isomer thereof, wherein 1 is selected from 1, 2, 3, 4, and5; M₁ is a bond or M′; R₄ is unsubstituted C₁₋₃ alkyl, or —(CH₂)_(n)Q,in which n is 2, 3, or 4, and Q is OH, —NHC(S)N(R)₂, —NHC(O)N(R)₂,—N(R)C(O)R, —N(R)S(O)₂R, —N(R)R₈, —NHC(═NR₉)N(R)₂, —NHC(═CHR₉)N(R)₂,—OC(O)N(R)₂, —N(R)C(O)OR, heteroaryl or heterocycloalkyl; M and M′ areindependently selectedfrom —C(O)O—, —OC(O)—, —C(O)N(R′)—, —P(O)(OR′)O—, —S—S—, an aryl group,and a heteroaryl group; and R₂ and R₃ are independently selected fromthe group consisting of H, C₁₋₁₄ alkyl, and C₂₋₁₄ alkenyl.

In certain embodiments, a subset of compounds of Formula (I) includesthose of Formula (IIa), (IIb), (IIc), or (IIe):

or a salt or isomer thereof, wherein R₄ is as described herein.

In certain embodiments, a subset of compounds of Formula (I) includesthose of Formula (IId):

or a salt or isomer thereof, wherein n is 2, 3, or 4; and m, R′, R″, andR₂ through R₆ are as described herein. For example, each of R₂ and R₃may be independently selected from the group consisting of C₅₋₁₄ alkyland C₅₋₁₄ alkenyl.

In another aspect, the disclosure features a nanoparticle compositionincluding a lipid component comprising a compound as described herein(e.g., a compound according to Formula (I), (IA), (II), (IIa), (IIb),(IIc), (IId) or (IIe)).

In yet another aspect, the disclosure features a pharmaceuticalcomposition comprising a nanoparticle composition according to thepreceding aspects and a pharmaceutically acceptable carrier. Forexample, the pharmaceutical composition is refrigerated or frozen forstorage and/or shipment (e.g., being stored at a temperature of 4° C. orlower, such as a temperature between about −150° C. and about 0° C. orbetween about −80° C. and about −20° C. (e.g., about −5° C., −10° C.,−15° C., −20° C., −25° C., −30° C., −40° C., −50° C., −60° C., −70° C.,−80° C., −90° C., −130° C. or −150° C.). For example, the pharmaceuticalcomposition is a solution that is refrigerated for storage and/orshipment at, for example, about −20° C., −30° C., −40° C., −50° C., −60°C., −70° C., or −80° C.

In another aspect, the disclosure provides a method of delivering atherapeutic and/or prophylactic (e.g., an mRNA) to a cell (e.g., amammalian cell). This method includes the step of administering to asubject (e.g., a mammal, such as a human) a nanoparticle compositionincluding (i) a lipid component including a phospholipid (such as apolyunsaturated lipid), a PEG lipid, a structural lipid, and a compoundof Formula (I), (IA), (II), (IIa), (IIb), (IIc), (IId) or (IIe) and (ii)a therapeutic and/or prophylactic, in which administering involvescontacting the cell with the nanoparticle composition, whereby thetherapeutic and/or prophylactic is delivered to the cell.

In another aspect, the disclosure provides a method of producing apolypeptide of interest in a cell (e.g., a mammalian cell). The methodincludes the step of contacting the cell with a nanoparticle compositionincluding (i) a lipid component including a phospholipid (such as apolyunsaturated lipid), a PEG lipid, a structural lipid, and a compoundof Formula (I), (IA), (II), (IIa), (IIb), (IIc), (IId) or (IIe) and (ii)an mRNA encoding the polypeptide of interest, whereby the mRNA iscapable of being translated in the cell to produce the polypeptide.

In another aspect, the disclosure provides a method of treating adisease or disorder in a mammal (e.g., a human) in need thereof. Themethod includes the step of administering to the mammal atherapeutically effective amount of a nanoparticle composition including(i) a lipid component including a phospholipid (such as apolyunsaturated lipid), a PEG lipid, a structural lipid, and a compoundof Formula (I), (IA), (II), (IIa), (IIb), (IIc), (IId) or (IIe) and (ii)a therapeutic and/or prophylactic (e.g., an mRNA). In some embodiments,the disease or disorder is characterized by dysfunctional or aberrantprotein or polypeptide activity. For example, the disease or disorder isselected from the group consisting of rare diseases, infectiousdiseases, cancer and proliferative diseases, genetic diseases (e.g.,cystic fibrosis), autoimmune diseases, diabetes, neurodegenerativediseases, cardio- and reno-vascular diseases, and metabolic diseases.

In another aspect, the disclosure provides a method of delivering (e.g.,specifically delivering) a therapeutic and/or prophylactic to amammalian organ (e.g., a liver, spleen, lung, or femur). This methodincludes the step of administering to a subject (e.g., a mammal) ananoparticle composition including (i) a lipid component including aphospholipid, a PEG lipid, a structural lipid, and a compound of Formula(I), (IA), (II), (IIa), (IIb), (IIc), (IId) or (IIe) and (ii) atherapeutic and/or prophylactic (e.g., an mRNA), in which administeringinvolves contacting the cell with the nanoparticle composition, wherebythe therapeutic and/or prophylactic is delivered to the target organ(e.g., a liver, spleen, lung, or femur).

In another aspect, the disclosure features a method for the enhanceddelivery of a therapeutic and/or prophylactic (e.g., an mRNA) to atarget tissue (e.g., a liver, spleen, lung, or femur). This methodincludes administering to a subject (e.g., a mammal) a nanoparticlecomposition, the composition including (i) a lipid component including acompound of Formula (I), (IA), (II), (IIa), (IIb), (IIc), (IId) or(IIe), a phospholipid, a structural lipid, and a PEG lipid; and (ii) atherapeutic and/or prophylactic, the administering including contactingthe target tissue with the nanoparticle composition, whereby thetherapeutic and/or prophylactic is delivered to the target tissue.

In yet another aspect, the disclosure features a method of loweringimmunogenicity comprising introducing the nanoparticle composition ofthe disclosure into cells, wherein the nanoparticle composition reducesthe induction of the cellular immune response of the cells to thenanoparticle composition, as compared to the induction of the cellularimmune response in cells induced by a reference composition whichcomprises a reference lipid instead of a compound of Formula (I), (IA),(II), (IIa), (IIb), (IIc), (IId) or (IIe). For example, the cellularimmune response is an innate immune response, an adaptive immuneresponse, or both.

The disclosure also includes methods of synthesizing a compound ofFormula (I), (IA), (II), (IIa), (IIb), (IIc), (IId) or (IIe) and methodsof making a nanoparticle composition including a lipid componentcomprising the compound of Formula (I), (IA), (II), (IIa), (IIb), (IIc),(IId) or (IIe).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the results of pretreating non-human primates withmethotrexate or dexamethasone prior to administration of a nanoparticlecomposition including MC3.

FIG. 2 shows the hEPO mRNA expression measured after intravenousadministration of various nanoparticle compositions at a 0.01 mpk dosewith 60 minutes infusion to naive cynomolgus monkeys.

FIGS. 3-6 respectively shows the results of hEPO expression measuredupon intravenous administration of various nanoparticle compositionsincluding Compounds 26, 18, 25, and MC3 to rat at various doses.

FIG. 7 shows the area under the curve (AUC) for nanoparticlecompositions including Compounds 18, 25, and 26 and MC3 at various dosesbetween 0.005 mpk and 2 mpk.

FIG. 8 shows the results of luciferase expression measured uponintramuscular administration of various nanoparticle compositionsincluding MC3, Compounds 168-170, and 173-175 to mice at 0.01 mpk atvarious time points: 3 hr (left block), 6 hr (middle block) and 24 hr(right block). The numbers 1-7 in this figure correspond to MC3,Compounds 168-170, and 173-175 respectively.

FIG. 9 shows the results of hEPO expression measured upon intramuscularadministration of various nanoparticle compositions including MC3,Compounds 18, 25, 30, 108-112, 60, and 122 to mice at 0.01 mpk atvarious time points: 3 hr (left block), 6 hr (middle block) and 24 hr(right block). The numbers 1-11 in this figure correspond to MC3,Compounds 18, 25, 30, 108-112, 60, and 122 respectively.

FIG. 10 shows the results of luciferase expression (total flux) measuredupon intravenous administration of various nanoparticle compositionsincluding MC3 or various compounds disclosed herein. The numbers 1-12 inthis figure correspond to Compound 18, MC3, Compounds 48-50, 54, 111,60, 75, 68, 66, 128, 65, 130, 133-135, 147, 96, and 151 respectively.

FIGS. 11A and 11B show the results of anti-HA (anti-hemagglutinin)antibody expression measured after intravenous administration of variousnanoparticle compositions including MC3 and Compound 18 at a 0.1 mpk(FIG. 11A) or 0.3 mpk (FIG. 11B) dose with 60 minutes infusion to naivecynomolgus monkeys.

DETAILED DESCRIPTION

The disclosure relates to novel lipids and lipid nanoparticlecompositions including a novel lipid. The disclosure also providesmethods of delivering a therapeutic and/or prophylactic to a mammaliancell, specifically delivering a therapeutic and/or prophylactic to amammalian organ, producing a polypeptide of interest in a mammaliancell, and treating a disease or disorder in a mammal in need thereof.For example, a method of producing a polypeptide of interest in a cellinvolves contacting a nanoparticle composition comprising an mRNA with amammalian cell, whereby the mRNA may be translated to produce thepolypeptide of interest. A method of delivering a therapeutic and/orprophylactic to a mammalian cell or organ may involve administration ofa nanoparticle composition including the therapeutic and/or prophylacticto a subject, in which the administration involves contacting the cellor organ with the composition, whereby the therapeutic and/orprophylactic is delivered to the cell or organ.

Lipids

The present disclosure provides lipids including a central amine moietyand at least one biodegradable group. The lipids described herein may beadvantageously used in lipid nanoparticle compositions for the deliveryof therapeutic and/or prophylactics to mammalian cells or organs. Forexample, the lipids described herein have little or no immunogenicity.For example, the lipid compound of any of Formula (I), (IA), (II),(IIa), (IIb), (IIc), (IId) or (IIe) has a lower immunogenicity ascompared to a reference lipid (e.g., MC3, KC2, or DLinDMA). For example,a formulation comprising a lipid disclosed herein and a therapeutic orprophylactic agent has an increased therapeutic index as compared to acorresponding formulation which comprise a reference lipid (e.g., MC3,KC2, or DLinDMA) and the same therapeutic or prophylactic agent.

In a first aspect of the invention, the compounds described herein areof Formula (I):

or salts or isomers thereof, wherein:

R₁ is selected from the group consisting of C₅₋₃₀ alkyl, C₅₋₂₀ alkenyl,—R*YR″, —YR″, and —R″M′R′;

R₂ and R₃ are independently selected from the group consisting of H,C₁₋₁₄ alkyl, C₂₋₁₄ alkenyl, —R*YR″, —YR″, and —R*OR″, or R₂ and R₃,together with the atom to which they are attached, form a heterocycle orcarbocycle;

R₄ is selected from the group consisting of a C₃₋₆ carbocycle,—(CH₂)_(n)Q, —(CH₂)_(n)CHQR, —CHQR, —CQ(R)₂, and unsubstituted C₁₋₆alkyl, where Q is selected from a carbocycle, heterocycle, —OR,—O(CH₂)_(n)N(R)₂, —C(O)OR, —OC(O)R, —CX₃, —CX₂H, —CXH₂, —CN, —N(R)₂,—C(O)N(R)₂, —N(R)C(O)R, —N(R)S(O)₂R, —N(R)C(O)N(R)₂, —N(R)C(S)N(R)₂,—N(R)R₈, —O(CH₂)_(n)OR, —N(R)C(═NR₉)N(R)₂, —N(R)C(═CHR₉)N(R)₂,—OC(O)N(R)₂, —N(R)C(O)OR, —N(OR)C(O)R, —N(OR)S(O)₂R, —N(OR)C(O)OR,—N(OR)C(O)N(R)₂, —N(OR)C(S)N(R)₂, —N(OR)C(═NR₉)N(R)₂,—N(OR)C(═CHR₉)N(R)₂, —C(═NR₉)N(R)₂, —C(═NR₉)R, —C(O)N(R)OR, and—C(R)N(R)₂C(O)OR, and each n is independently selected from 1, 2, 3, 4,and 5;

each R₅ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R₆ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

M and M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—,—N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—,—S(O)₂—, —S—S—, an aryl group, and a heteroaryl group;

R₇ is selected from the group consisting of C₁₋₃ alkyl, C₂₋₃ alkenyl,and H;

R₈ is selected from the group consisting of C₃₋₆ carbocycle andheterocycle;

R₉ is selected from the group consisting of H, CN, NO₂, C₁₋₆ alkyl, —OR,—S(O)₂R, —S(O)₂N(R)₂, C₂₋₆ alkenyl, C₃₋₆ carbocycle and heterocycle;

each R is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R′ is independently selected from the group consisting of C₁₋₁₈alkyl, C₂₋₁₈ alkenyl, —R*YR″, —YR″, and H;

each R″ is independently selected from the group consisting of C₃₋₁₄alkyl and C₃₋₁₄ alkenyl;

each R* is independently selected from the group consisting of C₁₋₁₂alkyl and C₂₋₁₂ alkenyl;

each Y is independently a C₃₋₆ carbocycle;

each X is independently selected from the group consisting of F, Cl, Br,and I; and

m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13; and wherein whenR₄ is —(CH₂)_(n)Q, —(CH₂)_(n)CHQR, —CHQR, or —CQ(R)₂, then (i) Q is not—N(R)₂ when n is 1, 2, 3, 4 or 5, or (ii) Q is not 5, 6, or 7-memberedheterocycloalkyl when n is 1 or 2.

In certain embodiments, a subset of compounds of Formula (I) includesthose of Formula (IA):

or a salt or isomer thereof, wherein 1 is selected from 1, 2, 3, 4, and5; m is selected from 5, 6, 7, 8, and 9; M₁ is a bond or M′; R₄ isunsubstituted C₁₋₃ alkyl, or —(CH₂)_(n)Q, in which Q is OH,—NHC(S)N(R)₂, —NHC(O)N(R)₂, —N(R)C(O)R, —N(R)S(O)₂R, —N(R)R₈,—NHC(═NR₉)N(R)₂, —NHC(═CHR₉)N(R)₂, —OC(O)N(R)₂, —N(R)C(O)OR, heteroarylor heterocycloalkyl; M and M′ are independently selectedfrom —C(O)O—, —OC(O)—, —C(O)N(R′)—, —P(O)(OR′)O—, —S—S—, an aryl group,and a heteroaryl group; and R₂ and R₃ are independently selected fromthe group consisting of H, C₁₋₁₄ alkyl, and C₂₋₁₄ alkenyl. For example,m is 5, 7, or 9. For example, Q is OH, —NHC(S)N(R)₂, or —NHC(O)N(R)₂.For example, Q is —N(R)C(O)R, or —N(R)S(O)₂R.

In certain embodiments, a subset of compounds of Formula (I) includesthose of Formula (II):

or a salt or isomer thereof, wherein 1 is selected from 1, 2, 3, 4, and5; M₁ is a bond or M′; R₄ is unsubstituted C₁₋₃ alkyl, or —(CH₂)_(n)Q,in which n is 2, 3, or 4, and Q is OH, —NHC(S)N(R)₂, —NHC(O)N(R)₂,—N(R)C(O)R, —N(R)S(O)₂R, —N(R)R₈, —NHC(═NR₉)N(R)₂, —NHC(═CHR₉)N(R)₂,—OC(O)N(R)₂, —N(R)C(O)OR, heteroaryl or heterocycloalkyl; M and M′ areindependently selectedfrom —C(O)O—, —OC(O)—, —C(O)N(R′)—, —P(O)(OR′)O—, —S—S—, an aryl group,and a heteroaryl group; and R₂ and R₃ are independently selected fromthe group consisting of H, C₁₋₁₄ alkyl, and C₂₋₁₄ alkenyl.

The compounds of any one of formula (I) or (IA) include one or more ofthe following features when applicable.

In some embodiments, M₁ is M′.

In some embodiments, M and M′ are independently —C(O)O— or —OC(O)—.

In some embodiments, at least one of M and M′ is —C(O)O— or —OC(O)—.

In some embodiments, M and M′ are independently —S—S—.

In some embodiments, at least one of M and M′ is —S—S.

In some embodiments, one of M and M′ is —C(O)O— or —OC(O)— and the otheris —S—S—. For example, M is —C(O)O— or —OC(O)— and M′ is —S—S— or M′ is—C(O)O— or —OC(O)— and M is —S—S—.

In some embodiments, l is 1, 3, or 5.

In some embodiments, R₄ is unsubstituted methyl or —(CH₂)_(n)Q, in whichQ is OH, —NHC(S)N(R)₂, —NHC(O)N(R)₂, —N(R)C(O)R, or —N(R)S(O)₂R.

In some embodiments, Q is OH.

In some embodiments, Q is —NHC(S)N(R)₂.

In some embodiments, Q is —NHC(O)N(R)₂.

In some embodiments, Q is —N(R)C(O)R.

In some embodiments, Q is —N(R)S(O)₂R.

In some embodiments, Q is —O(CH₂)_(n)N(R)₂.

In some embodiments, Q is —O(CH₂)_(n)OR.

In some embodiments, Q is —N(R)R₈.

In some embodiments, Q is —NHC(═NR₉)N(R)₂.

In some embodiments, Q is —NHC(═CHR₉)N(R)₂.

In some embodiments, Q is —OC(O)N(R)₂.

In some embodiments, Q is —N(R)C(O)OR.

In some embodiments, n is 2.

In some embodiments, n is 3.

In some embodiments, n is 4.

In some embodiments, M₁ is absent.

In some embodiments, R′ is C₁₋₁₈ alkyl, C₂₋₁₈ alkenyl, —R*YR″, or —YR″.

In some embodiments, R₂ and R₃ are independently C₃₋₁₄ alkyl or C₃₋₁₄alkenyl.

In one embodiment, the compounds of Formula (I) are of Formula (IIa),

or salts or isomers thereof, wherein R₄ is as described herein.

In another embodiment, the compounds of Formula (I) are of Formula(IIb),

or salts or isomers thereof, wherein R₄ is as described herein.

In another embodiment, the compounds of Formula (I) are of Formula (IIc)or (IIe):

or salts or isomers thereof, wherein R₄ is as described herein.

In a further embodiment, the compounds of Formula (I) are of Formula(IId),

or salts or isomers thereof, wherein n is 2, 3, or 4; and m, R′, R″, andR₂ through R₆ are as described herein. For example, each of R₂ and R₃may be independently selected from the group consisting of C₅₋₁₄ alkyland C₅₋₁₄ alkenyl.

The compounds of any one of formulae (I), (IA), (II), (IIa), (IIb),(IIc), (IId), and (IIe) include one or more of the following featureswhen applicable.

In some embodiments, R₄ is selected from the group consisting of a C₃₋₆carbocycle, —(CH₂)_(n)Q, —(CH₂)_(n)CHQR, —CHQR, and —CQ(R)₂, where Q isselected from a C₃₋₆ carbocycle, 5- to 14-membered aromatic ornon-aromatic heterocycle having one or more heteroatoms selected from N,O, S, and P, —OR, —O(CH₂)_(n)N(R)₂, —C(O)OR, —OC(O)R, —CX₃, —CX₂H,—CXH₂, —CN, —N(R)₂, —C(O)N(R)₂, —N(R)C(O)R, —N(R)S(O)₂R, —N(R)C(O)N(R)₂,—N(R)C(S)N(R)₂, and —C(R)N(R)₂C(O)OR, and each n is independentlyselected from 1, 2, 3, 4, and 5.

In another embodiment, R₄ is selected from the group consisting of aC₃₋₆ carbocycle, —(CH₂)_(n)Q, —(CH₂)_(n)CHQR, —CHQR, and —CQ(R)₂, whereQ is selected from a C₃₋₆ carbocycle, a 5- to 14-membered heteroarylhaving one or more heteroatoms selected from N, O, and S, —OR,—O(CH₂)_(n)N(R)₂, —C(O)OR, —OC(O)R, —CX₃, —CX₂H, —CXH₂, —CN, —C(O)N(R)₂,—N(R)C(O)R, —N(R)S(O)₂R, —N(R)C(O)N(R)₂, —N(R)C(S)N(R)₂,—C(R)N(R)₂C(O)OR, and a 5- to 14-membered heterocycloalkyl having one ormore heteroatoms selected from N, O, and S which is substituted with oneor more substituents selected from oxo (═O), OH, amino, and C₁₋₃ alkyl,and each n is independently selected from 1, 2, 3, 4, and 5.

In another embodiment, R₄ is selected from the group consisting of aC₃₋₆ carbocycle, —(CH₂)_(n)Q, —(CH₂)_(n)CHQR, —CHQR, and —CQ(R)₂, whereQ is selected from a C₃₋₆ carbocycle, a 5- to 14-membered heterocyclehaving one or more heteroatoms selected from N, O, and S, —OR,—O(CH₂)_(n)N(R)₂, —C(O)OR, —OC(O)R, —CX₃, —CX₂H, —CXH₂, —CN, —C(O)N(R)₂,—N(R)C(O)R, —N(R)S(O)₂R, —N(R)C(O)N(R)₂, —N(R)C(S)N(R)₂,—C(R)N(R)₂C(O)OR, and each n is independently selected from 1, 2, 3, 4,and 5; and when Q is a 5- to 14-membered heterocycle and (i) R₄ is—(CH₂)_(n)Q in which n is 1 or 2, or (ii) R₄ is —(CH₂)_(n)CHQR in whichn is 1, or (iii) R₄ is —CHQR, and —CQ(R)₂, then Q is either a 5- to14-membered heteroaryl or 8- to 14-membered heterocycloalkyl.

In another embodiment, R₄ is selected from the group consisting of aC₃₋₆ carbocycle, —(CH₂)_(n)Q, —(CH₂)_(n)CHQR, —CHQR, and —CQ(R)₂, whereQ is selected from a C₃₋₆ carbocycle, a 5- to 14-membered heteroarylhaving one or more heteroatoms selected from N, O, and S, —OR,—O(CH₂)_(n)N(R)₂, —C(O)OR, —OC(O)R, —CX₃, —CX₂H, —CXH₂, —CN, —C(O)N(R)₂,—N(R)C(O)R, —N(R)S(O)₂R, —N(R)C(O)N(R)₂, —N(R)C(S)N(R)₂,—C(R)N(R)₂C(O)OR, and each n is independently selected from 1, 2, 3, 4,and 5.

In another embodiment, R₄ is unsubstituted C₁₋₄ alkyl, e.g.,unsubstituted methyl.

In certain embodiments, the disclosure provides a compound having theFormula (I), wherein R₄ is —(CH₂)_(n)Q or —(CH₂)_(n)CHQR, where Q is—N(R)₂, and n is selected from 3, 4, and 5.

In certain embodiments, the disclosure provides a compound having theFormula (I), wherein R₄ is selected from the group consisting of—(CH₂)_(n)Q, —(CH₂)_(n)CHQR, —CHQR, and —CQ(R)₂, where Q is —N(R)₂, andn is selected from 1, 2, 3, 4, and 5.

In certain embodiments, the disclosure provides a compound having theFormula (I), wherein R₂ and R₃ are independently selected from the groupconsisting of C₂₋₁₄ alkyl, C₂₋₁₄ alkenyl, —R*YR″, —YR″, and —R*OR″, orR₂ and R₃, together with the atom to which they are attached, form aheterocycle or carbocycle, and R₄ is —(CH₂)_(n)Q or —(CH₂)_(n)CHQR,where Q is —N(R)₂, and n is selected from 3, 4, and 5.

In certain embodiments, R₂ and R₃ are independently selected from thegroup consisting of C₂₋₁₄ alkyl, C₂₋₁₄ alkenyl, —R*YR″, —YR″, and—R*OR″, or R₂ and R₃, together with the atom to which they are attached,form a heterocycle or carbocycle.

In some embodiments, R₁ is selected from the group consisting of C₅₋₂₀alkyl and C₅₋₂₀ alkenyl.

In other embodiments, R₁ is selected from the group consisting of—R*YR″, —YR″, and —R″M′R′.

In certain embodiments, R₁ is selected from —R*YR″ and —YR″. In someembodiments, Y is a cyclopropyl group. In some embodiments, R* is C₃alkyl or C₈ alkenyl. In certain embodiments, R″ is C₃₋₁₂ alkyl. Forexample, R″ may be C₃ alkyl. For example, R″ may be C₄₋₈ alkyl (e.g.,C₄, C₅, C₆, C₇, or C₈ alkyl).

In some embodiments, R₁ is C₅₋₂₀ alkyl. In some embodiments, R₁ is C₆alkyl. In some embodiments, R₁ is C₈ alkyl. In other embodiments, R₁ isC₉ alkyl. In certain embodiments, R₁ is C₁₄ alkyl. In other embodiments,R₁ is C₁₈ alkyl.

In some embodiments, R₁ is C₂₁₋₃₀ alkyl. In some embodiments, R₁ is C₂₆alkyl. In some embodiments, R₁ is C₂₈ alkyl. In certain embodiments, R₁is

In some embodiments, R₁ is C₅₋₂₀ alkenyl. In certain embodiments, R₁ isC₁₈ alkenyl. In some embodiments, R₁ is linoleyl.

In certain embodiments, R₁ is branched (e.g., decan-2-yl, undecan-3-yl,dodecan-4-yl, tridecan-5-yl, tetradecan-6-yl, 2-methylundecan-3-yl,2-methyldecan-2-yl, 3-methylundecan-3-yl, 4-methyldodecan-4-yl, orheptadeca-9-yl). In certain embodiments, R₁ is

In certain embodiments, R₁ is unsubstituted C₅₋₂₀ alkyl or C₅₋₂₀alkenyl. In certain embodiments, R′ is substituted C₅₋₂₀ alkyl or C₅₋₂₀alkenyl (e.g., substituted with a C₃₋₆ carbocycle such as1-cyclopropylnonyl or substituted with OH or alkoxy). For example, R₁ is

In other embodiments, R₁ is —R″M′R′.

In some embodiments, R′ is selected from —R*YR″ and —YR″. In someembodiments, Y is C₃₋₈ cycloalkyl. In some embodiments, Y is C₆₋₁₀ aryl.In some embodiments, Y is a cyclopropyl group. In some embodiments, Y isa cyclohexyl group. In certain embodiments, R* is C₁ alkyl.

In some embodiments, R″ is selected from the group consisting of C₃₋₁₂alkyl and C₃₋₁₂ alkenyl. In some embodiments, R″ adjacent to Y is C₁alkyl. In some embodiments, R″ adjacent to Y is C₄₋₉ alkyl (e.g., C₄,C₅, C₆, C₇ or C₈ or C₉ alkyl).

In some embodiments, R′ is selected from C₄ alkyl and C₄ alkenyl. Incertain embodiments, R′ is selected from C₅ alkyl and C₅ alkenyl. Insome embodiments, R′ is selected from C₆ alkyl and C₆ alkenyl. In someembodiments, R′ is selected from C₇ alkyl and C₇ alkenyl. In someembodiments, R′ is selected from C₉ alkyl and C₉ alkenyl.

In other embodiments, R′ is selected from C₁₁ alkyl and C₁₁ alkenyl. Inother embodiments, R′ is selected from C₁₂ alkyl, C₁₂ alkenyl, C₁₃alkyl, C₁₃ alkenyl, C₁₄ alkyl, C₁₄ alkenyl, C₁₅ alkyl, C₁₅ alkenyl, C₁₆alkyl, C₁₆ alkenyl, C₁₇ alkyl, C₁₇ alkenyl, C₁₈ alkyl, and C₁₈ alkenyl.In certain embodiments, R′ is branched (e.g., decan-2-yl, undecan-3-yl,dodecan-4-yl, tridecan-5-yl, tetradecan-6-yl, 2-methylundecan-3-yl,2-methyldecan-2-yl, 3-methylundecan-3-yl, 4-methyldodecan-4-yl orheptadeca-9-yl). In certain embodiments, R′ is

In certain embodiments, R′ is unsubstituted C₁₋₁₈ alkyl. In certainembodiments, R′ is substituted C₁₋₁₈ alkyl (e.g., C₁₋₁₅ alkylsubstituted with, e.g., an alkoxy such as methoxy, or a C₃₋₆ carbocyclesuch as 1-cyclopropylnonyl, or C(O)O-alkyl or OC(O)-alkyl such asC(O)OCH₃ or OC(O)CH₃). For example, R′ is

In some embodiments, R″ is selected from the group consisting of C₃₋₁₄alkyl and C₃₋₁₄ alkenyl. In some embodiments, R″ is C₃ alkyl, C₄ alkyl,C₅ alkyl, C₆ alkyl, C₇ alkyl, or C₈ alkyl. In some embodiments, R″ is C₉alkyl, C₁₀ alkyl, C₁₁ alkyl, C₁₂ alkyl, C₁₃ alkyl, or C₁₄ alkyl.

In some embodiments, M′ is —C(O)O—. In some embodiments, M′ is —OC(O)—.

In other embodiments, M′ is an aryl group or heteroaryl group. Forexample, M′ may be selected from the group consisting of phenyl,oxazole, and thiazole.

In some embodiments, M is —C(O)O—. In some embodiments, M is —OC(O)—. Insome embodiments, M is —C(O)N(R′)—. In some embodiments, M is—P(O)(OR′)O—.

In other embodiments, M is an aryl group or heteroaryl group. Forexample, M may be selected from the group consisting of phenyl, oxazole,and thiazole.

In some embodiments, M is the same as M′. In other embodiments, M isdifferent from M′.

In some embodiments, each R₅ is H. In certain such embodiments, each R₆is also H.

In some embodiments, R₇ is H. In other embodiments, R₇ is C₁₋₃ alkyl(e.g., methyl, ethyl, propyl, or i-propyl).

In some embodiments, R₂ and R₃ are independently C₅₋₁₄ alkyl or C₅₋₁₄alkenyl.

In some embodiments, R₂ and R₃ are the same. In some embodiments, R₂ andR₃ are C₈ alkyl. In certain embodiments, R₂ and R₃ are C₂ alkyl. Inother embodiments, R₂ and R₃ are C₃ alkyl. In some embodiments, R₂ andR₃ are C₄ alkyl. In certain embodiments, R₂ and R₃ are C₅ alkyl. Inother embodiments, R₂ and R₃ are C₆ alkyl. In some embodiments, R₂ andR₃ are C₇ alkyl.

In other embodiments, R₂ and R₃ are different. In certain embodiments,R₂ is C₈ alkyl. In some embodiments, R₃ is C₁₋₇ (e.g., C₁, C₂, C₃, C₄,C₅, C₆, or C₇ alkyl) or C₉ alkyl.

In some embodiments, R₇ and R₃ are H.

In certain embodiments, R₂ is H.

In some embodiments, m is 5, 7, or 9.

In some embodiments, R₄ is selected from —(CH₂)_(n)Q and —(CH₂)_(n)CHQR.

In some embodiments, Q is selected from the group consisting of —OR,—OH, —O(CH₂)_(n)N(R)₂, —OC(O)R, —CX₃, —CN, —N(R)C(O)R, —N(H)C(O)R,—N(R)S(O)₂R, —N(H)S(O)₂R, —N(R)C(O)N(R)₂, —N(H)C(O)N(R)₂,—N(H)C(O)N(H)(R), —N(R)C(S)N(R)₂, —N(H)C(S)N(R)₂, —N(H)C(S)N(H)(R),—C(R)N(R)₂C(O)OR, a carbocycle, and a heterocycle.

In certain embodiments, Q is —N(R)R₈, —O(CH₂)_(n)OR, —N(R)C(═NR₉)N(R)₂,—N(R)C(═CHR₉)N(R)₂, —OC(O)N(R)₂, or —N(R)C(O)OR.

In certain embodiments, Q is —N(OR)C(O)R, —N(OR)S(O)₂R, —N(OR)C(O)OR,—N(OR)C(O)N(R)₂, —N(OR)C(S)N(R)₂, —N(OR)C(═NR₉)N(R)₂, or—N(OR)C(═CHR₉)N(R)₂.

In certain embodiments, Q is thiourea or an isostere thereof, e.g.,

or —NHC(═NR₉)N(R)₂.

In certain embodiments, Q is —C(═NR₉)N(R)₂. For example, when Q is—C(═NR₉)N(R)₂, n is 4 or 5. For example, R₉ is —S(O)₂N(R)₂.

In certain embodiments, Q is —C(═NR₉)R or —C(O)N(R)OR, e.g.,—CH(═N—OCH₃), —C(O)NH—OH, —C(O)NH—OCH₃, —C(O)N(CH₃)—OH, or—C(O)N(CH₃)—OCH₃.

In certain embodiments, Q is —OH.

In certain embodiments, Q is a substituted or unsubstituted 5- to10-membered heteroaryl, e.g., Q is a triazole, an imidazole, apyrimidine, a purine, 2-amino-1,9-dihydro-6H-purin-6-one-9-yl (orguanin-9-yl), adenin-9-yl, cytosin-1-yl, or uracil-1-yl, each of whichis optionally substituted with one or more substituents selected fromalkyl, OH, alkoxy, -alkyl-OH, -alkyl-O-alkyl, and the substituent can befurther substituted. In certain embodiments, Q is a substituted 5- to14-membered heterocycloalkyl, e.g., substituted with one or moresubstituents selected from oxo (═O), OH, amino, mono- or di-alkylamino,and C₁₋₃ alkyl. For example, Q is 4-methylpiperazinyl,4-(4-methoxybenzyl)piperazinyl, isoindolin-2-yl-1,3-dione,pyrrolidin-1-yl-2,5-dione, or imidazolidin-3-yl-2,4-dione.

In certain embodiments, Q is —NHR₈, in which R₈ is a C₃-6 cycloalkyloptionally substituted with one or more substituents selected from oxo(═O), amino (NH₂), mono- or di-alkylamino, C₁₋₃ alkyl and halo. Forexample, R₈ is cyclobutenyl, e.g.,3-(dimethylamino)-cyclobut-3-ene-4-yl-1,2-dione.

In certain embodiments, Q is —NHR₈, in which R₈ is a heteroaryloptionally substituted with one or more substituents selected from amino(NH₂), mono- or di-alkylamino, C₁₋₃ alkyl and halo. For example, R₈ isthiazole or imidazole.

In certain embodiments, Q is —NHC(═NR₉)N(R)₂ in which R₉ is CN, C₁₋₆alkyl, NO₂, —S(O)₂N(R)₂, —OR, —S(O)₂R, or H. For example, Q is—NHC(═NR₉)N(CH₃)₂, —NHC(═NR₉)NHCH₃, —NHC(═NR₉)NH₂.

In certain embodiments, Q is —NHC(═CHR₉)N(R)₂, in which R₉ is NO₂, CN,C₁-6 alkyl, —S(O)₂N(R)₂, —OR, —S(O)₂R, or H. For example, Q is—NHC(═CHR₉)N(CH₃)₂, —NHC(═CHR₉)NHCH₃, or —NHC(═CHR₉)NH₂.

In certain embodiments, Q is —OC(O)N(R)₂, —N(R)C(O)OR, —N(OR)C(O)OR,such as —OC(O)NHCH₃, —N(OH)C(O)OCH₃, —N(OH)C(O)CH₃, —N(OCH₃)C(O)OCH₃,—N(OCH₃)C(O)CH₃, —N(OH)S(O)₂CH₃, or —NHC(O)OCH₃.

In certain embodiments, Q is an unsubstituted or substituted C₆₋₁₀ aryl(such as phenyl) or C₃₋₆ cycloalkyl.

In some embodiments, n is 1. In other embodiments, n is 2. In furtherembodiments, n is 3. In certain other embodiments, n is 4. For example,R₄ may be —(CH₂)₂OH. For example, R₄ may be —(CH₂)₃OH. For example, R₄may be —(CH₂)₄OH. For example, R₄ may be benzyl. For example, R₄ may be4-methoxybenzyl.

In some embodiments, R₄ is a C₃₋₆ carbocycle. In some embodiments, R₄ isa C₃₋₆ cycloalkyl. For example, R₄ may be cyclohexyl optionallysubstituted with e.g., OH, halo, C₁₋₆ alkyl, etc. For example, R₄ may be2-hydroxycyclohexyl.

In some embodiments, R is H.

In some embodiments, R is unsubstituted C₁₋₃ alkyl or unsubstituted C₂₋₃alkenyl. For example, R₄ may be —CH₂CH(OH)CH₃, —CH(CH₃)CH₂OH, or—CH₂CH(OH)CH₂CH₃.

In some embodiments, R is substituted C₁₋₃ alkyl, e.g., CH₂OH. Forexample, R₄ may be —CH₂CH(OH)CH₂OH, —(CH₂)₃NHC(O)CH₂OH,—(CH₂)₃NHC(O)CH₂OBn, —(CH₂)₂O(CH₂)₂OH, or —CH(CH₂OH)₂.

In some embodiments, R₄ is selected from any of the following groups:

In some embodiments, R₄ is selected from any of the following groups:

In some embodiments the compound of any of the formulae described hereinis suitable for making a nanoparticle composition for intramuscularadministration.

In some embodiments, R₂ and R₃, together with the atom to which they areattached, form a heterocycle or carbocycle. In some embodiments, R₂ andR₃, together with the atom to which they are attached, form a 5- to14-membered aromatic or non-aromatic heterocycle having one or moreheteroatoms selected from N, O, S, and P. In some embodiments, R₂ andR₃, together with the atom to which they are attached, form anoptionally substituted C₃₋₂₀ carbocycle (e.g., C₃₋₁₈ carbocycle, C₃₋₁₅carbocycle, C₃₋₁₂ carbocycle, or C₃₋₁₀ carbocycle), either aromatic ornon-aromatic. In some embodiments, R₂ and R₃, together with the atom towhich they are attached, form a C₃₋₆ carbocycle. In other embodiments,R₂ and R₃, together with the atom to which they are attached, form a C₆carbocycle, such as a cyclohexyl or phenyl group. In certainembodiments, the heterocycle or C₃₋₆ carbocycle is substituted with oneor more alkyl groups (e.g., at the same ring atom or at adjacent ornon-adjacent ring atoms). For example, R₂ and R₃, together with the atomto which they are attached, may form a cyclohexyl or phenyl groupbearing one or more C₅ alkyl substitutions. In certain embodiments, theheterocycle or C₃₋₆ carbocycle formed by R₂ and R₃, is substituted witha carbocycle groups. For example, R₂ and R₃, together with the atom towhich they are attached, may form a cyclohexyl or phenyl group that issubstituted with cyclohexyl. In some embodiments, R₂ and R₃, togetherwith the atom to which they are attached, form a C₇₋₁₅ carbocycle, suchas a cycloheptyl, cyclopentadecanyl, or naphthyl group.

In some embodiments, R₄ is selected from —(CH₂)_(n)Q and —(CH₂)_(n)CHQR.In some embodiments, Q is selected from the group consisting of —OR,—OH, —O(CH₂)_(n)N(R)₂, —OC(O)R, —CX₃, —CN, —N(R)C(O)R, —N(H)C(O)R,—N(R)S(O)₂R, —N(H)S(O)₂R, —N(R)C(O)N(R)₂, —N(H)C(O) N(R)₂,—N(H)C(O)N(H)(R), —N(R)C(S)N(R)₂, —N(H)C(S)N(R)₂, —N(H)C(S)N(H)(R), anda heterocycle. In other embodiments, Q is selected from the groupconsisting of an imidazole, a pyrimidine, and a purine.

In some embodiments, R₂ and R₃, together with the atom to which they areattached, form a heterocycle or carbocycle. In some embodiments, R₂ andR₃, together with the atom to which they are attached, form a C₃₋₆carbocycle, such as a phenyl group. In certain embodiments, theheterocycle or C₃₋₆ carbocycle is substituted with one or more alkylgroups (e.g., at the same ring atom or at adjacent or non-adjacent ringatoms). For example, R₂ and R₃, together with the atom to which they areattached, may form a phenyl group bearing one or more C₅ alkylsubstitutions.

In some embodiments, the compound of Formula (I) is selected from thegroup consisting of:

In further embodiments, the compound of Formula (I) is selected from thegroup consisting of:

In some embodiments, the compound of Formula (I) is selected from thegroup consisting of:

and salts and isomers thereof.

The central amine moiety of a lipid according to Formula (I), (IA),(II), (IIa), (IIb), (IIc), (IId) or (IIe) may be protonated at aphysiological pH. Thus, a lipid may have a positive or partial positivecharge at physiological pH. Such lipids may be referred to as cationicor ionizable (amino)lipids. Lipids may also be zwitterionic, i.e.,neutral molecules having both a positive and a negative charge.

As used herein, the term “alkyl” or “alkyl group” means a linear orbranched, saturated hydrocarbon including one or more carbon atoms(e.g., one, two, three, four, five, six, seven, eight, nine, ten,eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen,eighteen, nineteen, twenty, or more carbon atoms), which is optionallysubstituted. The notation “C₁₋₁₄ alkyl” means an optionally substitutedlinear or branched, saturated hydrocarbon including 1-14 carbon atoms.Unless otherwise specified, an alkyl group described herein refers toboth unsubstituted and substituted alkyl groups.

As used herein, the term “alkenyl” or “alkenyl group” means a linear orbranched hydrocarbon including two or more carbon atoms (e.g., two,three, four, five, six, seven, eight, nine, ten, eleven, twelve,thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen,twenty, or more carbon atoms) and at least one double bond, which isoptionally substituted. The notation “C₂₋₁₄ alkenyl” means an optionallysubstituted linear or branched hydrocarbon including 2-14 carbon atomsand at least one carbon-carbon double bond. An alkenyl group may includeone, two, three, four, or more carbon-carbon double bonds. For example,C₁₈ alkenyl may include one or more double bonds. A C₁₈ alkenyl groupincluding two double bonds may be a linoleyl group. Unless otherwisespecified, an alkenyl group described herein refers to bothunsubstituted and substituted alkenyl groups.

As used herein, the term “alkynyl” or “alkynyl group” means a linear orbranched hydrocarbon including two or more carbon atoms (e.g., two,three, four, five, six, seven, eight, nine, ten, eleven, twelve,thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen,twenty, or more carbon atoms) and at least one carbon-carbon triplebond, which is optionally substituted. The notation “C₂₋₁₄ alkynyl”means an optionally substituted linear or branched hydrocarbon including2-14 carbon atoms and at least one carbon-carbon triple bond. An alkynylgroup may include one, two, three, four, or more carbon-carbon triplebonds. For example, C₁₈ alkynyl may include one or more carbon-carbontriple bonds. Unless otherwise specified, an alkynyl group describedherein refers to both unsubstituted and substituted alkynyl groups.

As used herein, the term “carbocycle” or “carbocyclic group” means anoptionally substituted mono- or multi-cyclic system including one ormore rings of carbon atoms. Rings may be three, four, five, six, seven,eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen,seventeen, eighteen, nineteen, or twenty membered rings. The notation“C₃₋₆ carbocycle” means a carbocycle including a single ring having 3-6carbon atoms. Carbocycles may include one or more carbon-carbon doubleor triple bonds and may be non-aromatic or aromatic (e.g., cycloalkyl oraryl groups). Examples of carbocycles include cyclopropyl, cyclopentyl,cyclohexyl, phenyl, naphthyl, and 1,2-dihydronaphthyl groups. The term“cycloalkyl” as used herein means a non-aromatic carbocycle and may ormay not include any double or triple bond. Unless otherwise specified,carbocycles described herein refers to both unsubstituted andsubstituted carbocycle groups, i.e., optionally substituted carbocycles.

As used herein, the term “heterocycle” or “heterocyclic group” means anoptionally substituted mono- or multi-cyclic system including one ormore rings, where at least one ring includes at least one heteroatom.Heteroatoms may be, for example, nitrogen, oxygen, or sulfur atoms.Rings may be three, four, five, six, seven, eight, nine, ten, eleven,twelve, thirteen, or fourteen membered rings. Heterocycles may includeone or more double or triple bonds and may be non-aromatic or aromatic(e.g., heterocycloalkyl or heteroaryl groups). Examples of heterocyclesinclude imidazolyl, imidazolidinyl, oxazolyl, oxazolidinyl, thiazolyl,thiazolidinyl, pyrazolidinyl, pyrazolyl, isoxazolidinyl, isoxazolyl,isothiazolidinyl, isothiazolyl, morpholinyl, pyrrolyl, pyrrolidinyl,furyl, tetrahydrofuryl, thiophenyl, pyridinyl, piperidinyl, quinolyl,and isoquinolyl groups. The term “heterocycloalkyl” as used herein meansa non-aromatic heterocycle and may or may not include any double ortriple bond. Unless otherwise specified, heterocycles described hereinrefers to both unsubstituted and substituted heterocycle groups, i.e.,optionally substituted heterocycles.

As used herein, a “biodegradable group” is a group that may facilitatefaster metabolism of a lipid in a mammalian entity. A biodegradablegroup may be selected from the group consisting of, but is not limitedto, —C(O)O—, —OC(O)—, —C(O)N(R′)—, —N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—,—SC(S)—, —CH(OH)—, —P(O)(OR′)O—, —S(O)₂—, an aryl group, and aheteroaryl group. As used herein, an “aryl group” is an optionallysubstituted carbocyclic group including one or more aromatic rings.Examples of aryl groups include phenyl and naphthyl groups. As usedherein, a “heteroaryl group” is an optionally substituted heterocyclicgroup including one or more aromatic rings. Examples of heteroarylgroups include pyrrolyl, furyl, thiophenyl, imidazolyl, oxazolyl, andthiazolyl. Both aryl and heteroaryl groups may be optionallysubstituted. For example, M and M′ can be selected from the non-limitinggroup consisting of optionally substituted phenyl, oxazole, andthiazole. In the formulas herein, M and M′ can be independently selectedfrom the list of biodegradable groups above. Unless otherwise specified,aryl or heteroaryl groups described herein refers to both unsubstitutedand substituted groups, i.e., optionally substituted aryl or heteroarylgroups.

Alkyl, alkenyl, and cyclyl (e.g., carbocyclyl and heterocyclyl) groupsmay be optionally substituted unless otherwise specified. Optionalsubstituents may be selected from the group consisting of, but are notlimited to, a halogen atom (e.g., a chloride, bromide, fluoride, oriodide group), a carboxylic acid (e.g., —C(O)OH), an alcohol (e.g., ahydroxyl, —OH), an ester (e.g., —C(O)OR or —OC(O)R), an aldehyde (e.g.,—C(O)H), a carbonyl (e.g., —C(O)R, alternatively represented by C═O), anacyl halide (e.g., —C(O)X, in which X is a halide selected from bromide,fluoride, chloride, and iodide), a carbonate (e.g., —OC(O)OR), an alkoxy(e.g., —OR), an acetal (e.g., —C(OR)₂R″″, in which each OR are alkoxygroups that can be the same or different and R″″ is an alkyl or alkenylgroup), a phosphate (e.g., P(O)₄ ³⁻), a thiol (e.g., —SH), a sulfoxide(e.g., —S(O)R), a sulfinic acid (e.g., —S(O)OH), a sulfonic acid (e.g.,—S(O)₂OH), a thial (e.g., —C(S)H), a sulfate (e.g., S(O)₄ ²⁻), asulfonyl (e.g., —S(O)₂—), an amide (e.g., —C(O)NR₂, or —N(R)C(O)R), anazido (e.g., —N₃), a nitro (e.g., —NO₂), a cyano (e.g., —CN), anisocyano (e.g., —NC), an acyloxy (e.g., —OC(O)R), an amino (e.g., —NR₂,—NRH, or —NH₂), a carbamoyl (e.g., —OC(O)NR₂, —OC(O)NRH, or —OC(O)NH₂),a sulfonamide (e.g., —S(O)₂NR₂, —S(O)₂NRH, —S(O)₂NH₂, —N(R)S(O)₂R,—N(H)S(O)₂R, —N(R)S(O)₂H, or —N(H)S(O)₂H), an alkyl group, an alkenylgroup, and a cyclyl (e.g., carbocyclyl or heterocyclyl) group. In any ofthe preceding, R is an alkyl or alkenyl group, as defined herein. Insome embodiments, the substituent groups themselves may be furthersubstituted with, for example, one, two, three, four, five, or sixsubstituents as defined herein. For example, a C₁₋₆ alkyl group may befurther substituted with one, two, three, four, five, or sixsubstituents as described herein.

About, Approximately: As used herein, the terms “approximately” and“about,” as applied to one or more values of interest, refer to a valuethat is similar to a stated reference value. In certain embodiments, theterm “approximately” or “about” refers to a range of values that fallwithin 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%,8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greaterthan or less than) of the stated reference value unless otherwise statedor otherwise evident from the context (except where such number wouldexceed 100% of a possible value). For example, when used in the contextof an amount of a given compound in a lipid component of a nanoparticlecomposition, “about” may mean +/−10% of the recited value. For instance,a nanoparticle composition including a lipid component having about 40%of a given compound may include 30-50% of the compound.

As used herein, the term “compound,” is meant to include all isomers andisotopes of the structure depicted. “Isotopes” refers to atoms havingthe same atomic number but different mass numbers resulting from adifferent number of neutrons in the nuclei. For example, isotopes ofhydrogen include tritium and deuterium. Further, a compound, salt, orcomplex of the present disclosure can be prepared in combination withsolvent or water molecules to form solvates and hydrates by routinemethods.

As used herein, the term “contacting” means establishing a physicalconnection between two or more entities. For example, contacting amammalian cell with a nanoparticle composition means that the mammaliancell and a nanoparticle are made to share a physical connection. Methodsof contacting cells with external entities both in vivo and ex vivo arewell known in the biological arts. For example, contacting ananoparticle composition and a mammalian cell disposed within a mammalmay be performed by varied routes of administration (e.g., intravenous,intramuscular, intradermal, and subcutaneous) and may involve variedamounts of nanoparticle compositions. Moreover, more than one mammaliancell may be contacted by a nanoparticle composition.

As used herein, the term “delivering” means providing an entity to adestination. For example, delivering a therapeutic and/or prophylacticto a subject may involve administering a nanoparticle compositionincluding the therapeutic and/or prophylactic to the subject (e.g., byan intravenous, intramuscular, intradermal, or subcutaneous route).Administration of a nanoparticle composition to a mammal or mammaliancell may involve contacting one or more cells with the nanoparticlecomposition.

As used herein, the term “enhanced delivery” means delivery of more(e.g., at least 1.5 fold more, at least 2-fold more, at least 3-foldmore, at least 4-fold more, at least 5-fold more, at least 6-fold more,at least 7-fold more, at least 8-fold more, at least 9-fold more, atleast 10-fold more) of a therapeutic and/or prophylactic by ananoparticle to a target tissue of interest (e.g., mammalian liver)compared to the level of delivery of a therapeutic and/or prophylacticby a control nanoparticle to a target tissue of interest (e.g., MC3,KC2, or DLinDMA). The level of delivery of a nanoparticle to aparticular tissue may be measured by comparing the amount of proteinproduced in a tissue to the weight of said tissue, comparing the amountof therapeutic and/or prophylactic in a tissue to the weight of saidtissue, comparing the amount of protein produced in a tissue to theamount of total protein in said tissue, or comparing the amount oftherapeutic and/or prophylactic in a tissue to the amount of totaltherapeutic and/or prophylactic in said tissue. It will be understoodthat the enhanced delivery of a nanoparticle to a target tissue need notbe determined in a subject being treated, it may be determined in asurrogate such as an animal model (e.g., a rat model). In certainembodiments, a nanoparticle composition including a compound accordingto Formula (I), (IA), (II), (IIa), (IIb), (IIc), (IId) or (IIe) hassubstantively the same level of delivery enhancement regardless ofadministration routes. For example, certain compounds disclosed hereinexhibit similar delivery enhancement when they are used for delivering atherapeutic and/or prophylactic either intravenously or intramuscularly.In other embodiments, certain compounds disclosed herein (e.g., acompound of Formula (IA) or (II), such as Compound 18, 25, 30, 60,108-112, or 122) exhibit a higher level of delivery enhancement whenthey are used for delivering a therapeutic and/or prophylacticintramuscularly than intravenously.

As used herein, the term “specific delivery,” “specifically deliver,” or“specifically delivering” means delivery of more (e.g., at least 1.5fold more, at least 2-fold more, at least 3-fold more, at least 4-foldmore, at least 5-fold more, at least 6-fold more, at least 7-fold more,at least 8-fold more, at least 9-fold more, at least 10-fold more) of atherapeutic and/or prophylactic by a nanoparticle to a target tissue ofinterest (e.g., mammalian liver) compared to an off-target tissue (e.g.,mammalian spleen). The level of delivery of a nanoparticle to aparticular tissue may be measured by comparing the amount of proteinproduced in a tissue to the weight of said tissue, comparing the amountof therapeutic and/or prophylactic in a tissue to the weight of saidtissue, comparing the amount of protein produced in a tissue to theamount of total protein in said tissue, or comparing the amount oftherapeutic and/or prophylactic in a tissue to the amount of totaltherapeutic and/or prophylactic in said tissue. For example, forrenovascular targeting, a therapeutic and/or prophylactic isspecifically provided to a mammalian kidney as compared to the liver andspleen if 1.5, 2-fold, 3-fold, 5-fold, 10-fold, 15 fold, or 20 fold moretherapeutic and/or prophylactic per 1 g of tissue is delivered to akidney compared to that delivered to the liver or spleen followingsystemic administration of the therapeutic and/or prophylactic. It willbe understood that the ability of a nanoparticle to specifically deliverto a target tissue need not be determined in a subject being treated, itmay be determined in a surrogate such as an animal model (e.g., a ratmodel).

As used herein, “encapsulation efficiency” refers to the amount of atherapeutic and/or prophylactic that becomes part of a nanoparticlecomposition, relative to the initial total amount of therapeutic and/orprophylactic used in the preparation of a nanoparticle composition. Forexample, if 97 mg of therapeutic and/or prophylactic are encapsulated ina nanoparticle composition out of a total 100 mg of therapeutic and/orprophylactic initially provided to the composition, the encapsulationefficiency may be given as 97%. As used herein, “encapsulation” mayrefer to complete, substantial, or partial enclosure, confinement,surrounding, or encasement.

As used herein, “expression” of a nucleic acid sequence refers totranslation of an mRNA into a polypeptide or protein and/orpost-translational modification of a polypeptide or protein.

As used herein, the term “in vitro” refers to events that occur in anartificial environment, e.g., in a test tube or reaction vessel, in cellculture, in a Petri dish, etc., rather than within an organism (e.g.,animal, plant, or microbe).

As used herein, the term “in vivo” refers to events that occur within anorganism (e.g., animal, plant, or microbe or cell or tissue thereof).

As used herein, the term “ex vivo” refers to events that occur outsideof an organism (e.g., animal, plant, or microbe or cell or tissuethereof). Ex vivo events may take place in an environment minimallyaltered from a natural (e.g., in vivo) environment.

As used herein, the term “isomer” means any geometric isomer, tautomer,zwitterion, stereoisomer, enantiomer, or diastereomer of a compound.Compounds may include one or more chiral centers and/or double bonds andmay thus exist as stereoisomers, such as double-bond isomers (i.e.,geometric E/Z isomers) or diastereomers (e.g., enantiomers (i.e., (+) or(−)) or cis/trans isomers). The present disclosure encompasses any andall isomers of the compounds described herein, including stereomericallypure forms (e.g., geometrically pure, enantiomerically pure, ordiastereomerically pure) and enantiomeric and stereoisomeric mixtures,e.g., racemates. Enantiomeric and stereomeric mixtures of compounds andmeans of resolving them into their component enantiomers orstereoisomers are well-known.

As used herein, a “lipid component” is that component of a nanoparticlecomposition that includes one or more lipids. For example, the lipidcomponent may include one or more cationic/ionizable, PEGylated,structural, or other lipids, such as phospholipids.

As used herein, a “linker” is a moiety connecting two moieties, forexample, the connection between two nucleosides of a cap species. Alinker may include one or more groups including but not limited tophosphate groups (e.g., phosphates, boranophosphates, thiophosphates,selenophosphates, and phosphonates), alkyl groups, amidates, orglycerols. For example, two nucleosides of a cap analog may be linked attheir 5′ positions by a triphosphate group or by a chain including twophosphate moieties and a boranophosphate moiety.

As used herein, “methods of administration” may include intravenous,intramuscular, intradermal, subcutaneous, or other methods of deliveringa composition to a subject. A method of administration may be selectedto target delivery (e.g., to specifically deliver) to a specific regionor system of a body.

As used herein, “modified” means non-natural. For example, an RNA may bea modified RNA. That is, an RNA may include one or more nucleobases,nucleosides, nucleotides, or linkers that are non-naturally occurring. A“modified” species may also be referred to herein as an “altered”species. Species may be modified or altered chemically, structurally, orfunctionally. For example, a modified nucleobase species may include oneor more substitutions that are not naturally occurring.

As used herein, the “N:P ratio” is the molar ratio of ionizable (in thephysiological pH range) nitrogen atoms in a lipid to phosphate groups inan RNA, e.g., in a nanoparticle composition including a lipid componentand an RNA.

As used herein, a “nanoparticle composition” is a composition comprisingone or more lipids. Nanoparticle compositions are typically sized on theorder of micrometers or smaller and may include a lipid bilayer.Nanoparticle compositions encompass lipid nanoparticles (LNPs),liposomes (e.g., lipid vesicles), and lipoplexes. For example, ananoparticle composition may be a liposome having a lipid bilayer with adiameter of 500 nm or less.

As used herein, “naturally occurring” means existing in nature withoutartificial aid.

As used herein, “patient” refers to a subject who may seek or be in needof treatment, requires treatment, is receiving treatment, will receivetreatment, or a subject who is under care by a trained professional fora particular disease or condition.

As used herein, a “PEG lipid” or “PEGylated lipid” refers to a lipidcomprising a polyethylene glycol component.

The phrase “pharmaceutically acceptable” is used herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically acceptable excipient,” as used herein,refers to any ingredient other than the compounds described herein (forexample, a vehicle capable of suspending, complexing, or dissolving theactive compound) and having the properties of being substantiallynontoxic and non-inflammatory in a patient. Excipients may include, forexample: anti-adherents, antioxidants, binders, coatings, compressionaids, disintegrants, dyes (colors), emollients, emulsifiers, fillers(diluents), film formers or coatings, flavors, fragrances, glidants(flow enhancers), lubricants, preservatives, printing inks, sorbents,suspending or dispersing agents, sweeteners, and waters of hydration.Exemplary excipients include, but are not limited to: butylatedhydroxytoluene (BHT), calcium carbonate, calcium phosphate (dibasic),calcium stearate, croscarmellose, crosslinked polyvinyl pyrrolidone,citric acid, crospovidone, cysteine, ethylcellulose, gelatin,hydroxypropyl cellulose, hydroxypropyl methylcellulose, lactose,magnesium stearate, maltitol, mannitol, methionine, methylcellulose,methyl paraben, microcrystalline cellulose, polyethylene glycol,polyvinyl pyrrolidone, povidone, pregelatinized starch, propyl paraben,retinyl palmitate, shellac, silicon dioxide, sodium carboxymethylcellulose, sodium citrate, sodium starch glycolate, sorbitol, starch(corn), stearic acid, sucrose, talc, titanium dioxide, vitamin A,vitamin E (alpha-tocopherol), vitamin C, xylitol, and other speciesdisclosed herein.

In the present specification, the structural formula of the compoundrepresents a certain isomer for convenience in some cases, but thepresent disclosure includes all isomers, such as geometrical isomers,optical isomers based on an asymmetrical carbon, stereoisomers,tautomers, and the like, it being understood that not all isomers mayhave the same level of activity. In addition, a crystal polymorphism maybe present for the compounds represented by the formula. It is notedthat any crystal form, crystal form mixture, or anhydride or hydratethereof is included in the scope of the present disclosure.

The term “crystal polymorphs”, “polymorphs” or “crystal forms” meanscrystal structures in which a compound (or a salt or solvate thereof)can crystallize in different crystal packing arrangements, all of whichhave the same elemental composition. Different crystal forms usuallyhave different X-ray diffraction patterns, infrared spectral, meltingpoints, density hardness, crystal shape, optical and electricalproperties, stability and solubility. Recrystallization solvent, rate ofcrystallization, storage temperature, and other factors may cause onecrystal form to dominate. Crystal polymorphs of the compounds can beprepared by crystallization under different conditions.

Compositions may also include salts of one or more compounds. Salts maybe pharmaceutically acceptable salts. As used herein, “pharmaceuticallyacceptable salts” refers to derivatives of the disclosed compoundswherein the parent compound is altered by converting an existing acid orbase moiety to its salt form (e.g., by reacting a free base group with asuitable organic acid). Examples of pharmaceutically acceptable saltsinclude, but are not limited to, mineral or organic acid salts of basicresidues such as amines; alkali or organic salts of acidic residues suchas carboxylic acids; and the like. Representative acid addition saltsinclude acetate, adipate, alginate, ascorbate, aspartate,benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate,camphorsulfonate, citrate, cyclopentanepropionate, digluconate,dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate,glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide,hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate,lactate, laurate, lauryl sulfate, malate, maleate, malonate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate,oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate,phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate,tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate salts,and the like. Representative alkali or alkaline earth metal saltsinclude sodium, lithium, potassium, calcium, magnesium, and the like, aswell as nontoxic ammonium, quaternary ammonium, and amine cations,including, but not limited to ammonium, tetramethylammonium,tetraethylammonium, methylamine, dimethylamine, trimethylamine,triethylamine, ethylamine, and the like. The pharmaceutically acceptablesalts of the present disclosure include the conventional non-toxic saltsof the parent compound formed, for example, from non-toxic inorganic ororganic acids. The pharmaceutically acceptable salts of the presentdisclosure can be synthesized from the parent compound which contains abasic or acidic moiety by conventional chemical methods. Generally, suchsalts can be prepared by reacting the free acid or base forms of thesecompounds with a stoichiometric amount of the appropriate base or acidin water or in an organic solvent, or in a mixture of the two;generally, nonaqueous media like ether, ethyl acetate, ethanol,isopropanol, or acetonitrile are preferred. Lists of suitable salts arefound in Remington's Pharmaceutical Sciences, 17^(th) ed., MackPublishing Company, Easton, Pa., 1985, p. 1418, Pharmaceutical Salts:Properties, Selection, and Use, P. H. Stahl and C. G. Wermuth (eds.),Wiley-VCH, 2008, and Berge et al., Journal of Pharmaceutical Science,66, 1-19 (1977), each of which is incorporated herein by reference inits entirety.

As used herein, a “phospholipid” is a lipid that includes a phosphatemoiety and one or more carbon chains, such as unsaturated fatty acidchains. A phospholipid may include one or more multiple (e.g., double ortriple) bonds (e.g., one or more unsaturations). Particularphospholipids may facilitate fusion to a membrane. For example, acationic phospholipid may interact with one or more negatively chargedphospholipids of a membrane (e.g., a cellular or intracellularmembrane). Fusion of a phospholipid to a membrane may allow one or moreelements of a lipid-containing composition to pass through the membranepermitting, e.g., delivery of the one or more elements to a cell.

As used herein, the “polydispersity index” is a ratio that describes thehomogeneity of the particle size distribution of a system. A smallvalue, e.g., less than 0.3, indicates a narrow particle sizedistribution.

As used herein, the term “polypeptide” or “polypeptide of interest”refers to a polymer of amino acid residues typically joined by peptidebonds that can be produced naturally (e.g., isolated or purified) orsynthetically.

As used herein, an “RNA” refers to a ribonucleic acid that may benaturally or non-naturally occurring. For example, an RNA may includemodified and/or non-naturally occurring components such as one or morenucleobases, nucleosides, nucleotides, or linkers. An RNA may include acap structure, a chain terminating nucleoside, a stem loop, a polyAsequence, and/or a polyadenylation signal. An RNA may have a nucleotidesequence encoding a polypeptide of interest. For example, an RNA may bea messenger RNA (mRNA). Translation of an mRNA encoding a particularpolypeptide, for example, in vivo translation of an mRNA inside amammalian cell, may produce the encoded polypeptide. RNAs may beselected from the non-liming group consisting of small interfering RNA(siRNA), asymmetrical interfering RNA (aiRNA), microRNA (miRNA),Dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), mRNA, andmixtures thereof.

As used herein, a “single unit dose” is a dose of any therapeuticadministered in one dose/at one time/single route/single point ofcontact, i.e., single administration event.

As used herein, a “split dose” is the division of single unit dose ortotal daily dose into two or more doses.

As used herein, a “total daily dose” is an amount given or prescribed in24 hour period. It may be administered as a single unit dose.

As used herein, “size” or “mean size” in the context of nanoparticlecompositions refers to the mean diameter of a nanoparticle composition.

As used herein, the term “subject” or “patient” refers to any organismto which a composition in accordance with the disclosure may beadministered, e.g., for experimental, diagnostic, prophylactic, and/ortherapeutic purposes. Typical subjects include animals (e.g., mammalssuch as mice, rats, rabbits, non-human primates, and humans) and/orplants.

As used herein, “targeted cells” refers to any one or more cells ofinterest. The cells may be found in vitro, in vivo, in situ, or in thetissue or organ of an organism. The organism may be an animal,preferably a mammal, more preferably a human and most preferably apatient.

As used herein “target tissue” refers to any one or more tissue types ofinterest in which the delivery of a therapeutic and/or prophylacticwould result in a desired biological and/or pharmacological effect.Examples of target tissues of interest include specific tissues, organs,and systems or groups thereof. In particular applications, a targettissue may be a kidney, a lung, a spleen, vascular endothelium invessels (e.g., intra-coronary or intra-femoral), or tumor tissue (e.g.,via intratumoral injection). An “off-target tissue” refers to any one ormore tissue types in which the expression of the encoded protein doesnot result in a desired biological and/or pharmacological effect. Inparticular applications, off-target tissues may include the liver andthe spleen.

The term “therapeutic agent” or “prophylactic agent” refers to any agentthat, when administered to a subject, has a therapeutic, diagnostic,and/or prophylactic effect and/or elicits a desired biological and/orpharmacological effect. Therapeutic agents are also referred to as“actives” or “active agents.” Such agents include, but are not limitedto, cytotoxins, radioactive ions, chemotherapeutic agents, smallmolecule drugs, proteins, and nucleic acids.

As used herein, the term “therapeutically effective amount” means anamount of an agent to be delivered (e.g., nucleic acid, drug,composition, therapeutic agent, diagnostic agent, prophylactic agent,etc.) that is sufficient, when administered to a subject suffering fromor susceptible to an infection, disease, disorder, and/or condition, totreat, improve symptoms of, diagnose, prevent, and/or delay the onset ofthe infection, disease, disorder, and/or condition.

As used herein, “transfection” refers to the introduction of a species(e.g., an RNA) into a cell. Transfection may occur, for example, invitro, ex vivo, or in vivo.

As used herein, the term “treating” refers to partially or completelyalleviating, ameliorating, improving, relieving, delaying onset of,inhibiting progression of, reducing severity of, and/or reducingincidence of one or more symptoms or features of a particular infection,disease, disorder, and/or condition. For example, “treating” cancer mayrefer to inhibiting survival, growth, and/or spread of a tumor.Treatment may be administered to a subject who does not exhibit signs ofa disease, disorder, and/or condition and/or to a subject who exhibitsonly early signs of a disease, disorder, and/or condition for thepurpose of decreasing the risk of developing pathology associated withthe disease, disorder, and/or condition.

As used herein, the “zeta potential” is the electrokinetic potential ofa lipid, e.g., in a particle composition.

Nanoparticle Compositions

The disclosure also features nanoparticle compositions comprising alipid component comprising a compound according to Formula (I), (IA),(II), (IIa), (IIb), (IIc), (IId) or (IIe) as described herein.

In some embodiments, the largest dimension of a nanoparticle compositionis 1 μm or shorter (e.g., 1 μm, 900 nm, 800 nm, 700 nm, 600 nm, 500 nm,400 nm, 300 nm, 200 nm, 175 nm, 150 nm, 125 nm, 100 nm, 75 nm, 50 nm, orshorter), e.g., when measured by dynamic light scattering (DLS),transmission electron microscopy, scanning electron microscopy, oranother method. Nanoparticle compositions include, for example, lipidnanoparticles (LNPs), liposomes, lipid vesicles, and lipoplexes. In someembodiments, nanoparticle compositions are vesicles including one ormore lipid bilayers. In certain embodiments, a nanoparticle compositionincludes two or more concentric bilayers separated by aqueouscompartments. Lipid bilayers may be functionalized and/or crosslinked toone another. Lipid bilayers may include one or more ligands, proteins,or channels.

Nanoparticle compositions comprise a lipid component including at leastone compound according to Formula (I), (IA), (II), (IIa), (IIb), (IIc),(IId) or (IIe). For example, the lipid component of a nanoparticlecomposition may include one or more of Compounds 1-147. Nanoparticlecompositions may also include a variety of other components. Forexample, the lipid component of a nanoparticle composition may includeone or more other lipids in addition to a lipid according to Formula(I), (IA), (II), (IIa), (IIb), (IIc), (IId) or (IIe).

Cationic/Ionizable Lipids

A nanoparticle composition may include one or more cationic and/orionizable lipids (e.g., lipids that may have a positive or partialpositive charge at physiological pH) in addition to a lipid according toFormula (I), (IA), (II), (IIa), (IIb), (IIc), (IId) or (IIe). Cationicand/or ionizable lipids may be selected from the non-limiting groupconsisting of3-(didodecylamino)-N1,N1,4-tridodecyl-1-piperazineethanamine (KL10),N1-[2-(didodecylamino)ethyl]-N1,N4,N4-tridodecyl-1,4-piperazinediethanamine(KL22), 14,25-ditridecyl-15,18,21,24-tetraaza-octatriacontane (KL25),1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA),2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA),heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate(DLin-MC3-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane(DLin-KC2-DMA), 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA),2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine(Octyl-CLinDMA),(2R)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine(Octyl-CLinDMA (2R)), and(2S)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine(Octyl-CLinDMA (2S)). In addition to these, a cationic lipid may also bea lipid including a cyclic amine group.

PEG Lipids

The lipid component of a nanoparticle composition may include one ormore PEG or PEG-modified lipids. Such species may be alternatelyreferred to as PEGylated lipids. A PEG lipid is a lipid modified withpolyethylene glycol. A PEG lipid may be selected from the non-limitinggroup consisting of PEG-modified phosphatidylethanolamines, PEG-modifiedphosphatidic acids, PEG-modified ceramides, PEG-modified dialkylamines,PEG-modified diacylglycerols, PEG-modified dialkylglycerols, andmixtures thereof. For example, a PEG lipid may be PEG-c-DOMG, PEG-DMG,PEG-DLPE, PEG-DMPE, PEG-DPPC, or a PEG-DSPE lipid.

Structural Lipids

The lipid component of a nanoparticle composition may include one ormore structural lipids. Structural lipids can be selected from the groupconsisting of, but are not limited to, cholesterol, fecosterol,sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol,tomatidine, tomatine, ursolic acid, alpha-tocopherol, and mixturesthereof. In some embodiments, the structural lipid is cholesterol. Insome embodiments, the structural lipid includes cholesterol and acorticosteroid (such as prednisolone, dexamethasone, prednisone, andhydrocortisone), or a combination thereof.

Phospholipids

The lipid component of a nanoparticle composition may include one ormore phospholipids, such as one or more (poly)unsaturated lipids.Phospholipids may assemble into one or more lipid bilayers. In general,phospholipids may include a phospholipid moiety and one or more fattyacid moieties. For example, a phospholipid may be a lipid according toFormula (III):

in which R_(p) represents a phospholipid moiety and R₁ and R₂ representfatty acid moieties with or without unsaturation that may be the same ordifferent. A phospholipid moiety may be selected from the non-limitinggroup consisting of phosphatidyl choline, phosphatidyl ethanolamine,phosphatidyl glycerol, phosphatidyl serine, phosphatidic acid,2-lysophosphatidyl choline, and a sphingomyelin. A fatty acid moiety maybe selected from the non-limiting group consisting of lauric acid,myristic acid, myristoleic acid, palmitic acid, palmitoleic acid,stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucicacid, phytanoic acid, arachidic acid, arachidonic acid, eicosapentaenoicacid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid.Non-natural species including natural species with modifications andsubstitutions including branching, oxidation, cyclization, and alkynesare also contemplated. For example, a phospholipid may be functionalizedwith or cross-linked to one or more alkynes (e.g., an alkenyl group inwhich one or more double bonds is replaced with a triple bond). Underappropriate reaction conditions, an alkyne group may undergo acopper-catalyzed cycloaddition upon exposure to an azide. Such reactionsmay be useful in functionalizing a lipid bilayer of a nanoparticlecomposition to facilitate membrane permeation or cellular recognition orin conjugating a nanoparticle composition to a useful component such asa targeting or imaging moiety (e.g., a dye).

Phospholipids useful in the compositions and methods may be selectedfrom the non-limiting group consisting of1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC),1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE),1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC),1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC),1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC),1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC),1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC),1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC),1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine(OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC),1,2-dilinolenoyl-sn-glycero-3-phosphocholine,1,2-diarachidonoyl-sn-glycero-3-phosphocholine,1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine,1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE),1,2-distearoyl-sn-glycero-3-phosphoethanolamine,1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine,1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine,1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine,1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine,1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG),and sphingomyelin. In some embodiments, a nanoparticle compositionincludes DSPC. In certain embodiments, a nanoparticle compositionincludes DOPE. In some embodiments, a nanoparticle composition includesboth DSPC and DOPE.

Adjuvants

In some embodiments, a nanoparticle composition that includes one ormore lipids described herein may further include one or more adjuvants,e.g., Glucopyranosyl Lipid Adjuvant (GLA), CpG oligodeoxynucleotides(e.g., Class A or B), poly(I:C), aluminum hydroxide, and Pam3CSK4.

Therapeutic Agents

Nanoparticle compositions may include one or more therapeutic and/orprophylactics. The disclosure features methods of delivering atherapeutic and/or prophylactic to a mammalian cell or organ, producinga polypeptide of interest in a mammalian cell, and treating a disease ordisorder in a mammal in need thereof comprising administering to amammal and/or contacting a mammalian cell with a nanoparticlecomposition including a therapeutic and/or prophylactic.

Therapeutic and/or prophylactics include biologically active substancesand are alternately referred to as “active agents.” A therapeutic and/orprophylactic may be a substance that, once delivered to a cell or organ,brings about a desirable change in the cell, organ, or other bodilytissue or system. Such species may be useful in the treatment of one ormore diseases, disorders, or conditions. In some embodiments, atherapeutic and/or prophylactic is a small molecule drug useful in thetreatment of a particular disease, disorder, or condition. Examples ofdrugs useful in the nanoparticle compositions include, but are notlimited to, antineoplastic agents (e.g., vincristine, doxorubicin,mitoxantrone, camptothecin, cisplatin, bleomycin, cyclophosphamide,methotrexate, and streptozotocin), antitumor agents (e.g., actinomycinD, vincristine, vinblastine, cystine arabinoside, anthracyclines,alkylative agents, platinum compounds, antimetabolites, and nucleosideanalogs, such as methotrexate and purine and pyrimidine analogs),anti-infective agents, local anesthetics (e.g., dibucaine andchlorpromazine), beta-adrenergic blockers (e.g., propranolol, timolol,and labetolol), antihypertensive agents (e.g., clonidine andhydralazine), anti-depressants (e.g., imipramine, amitriptyline, anddoxepim), anti-conversants (e.g., phenytoin), antihistamines (e.g.,diphenhydramine, chlorphenirimine, and promethazine),antibiotic/antibacterial agents (e.g., gentamycin, ciprofloxacin, andcefoxitin), antifungal agents (e.g., miconazole, terconazole, econazole,isoconazole, butaconazole, clotrimazole, itraconazole, nystatin,naftifine, and amphotericin B), antiparasitic agents, hormones, hormoneantagonists, immunomodulators, neurotransmitter antagonists,antiglaucoma agents, vitamins, narcotics, and imaging agents.

In some embodiments, a therapeutic and/or prophylactic is a cytotoxin, aradioactive ion, a chemotherapeutic, a vaccine, a compound that elicitsan immune response, and/or another therapeutic and/or prophylactic. Acytotoxin or cytotoxic agent includes any agent that may be detrimentalto cells. Examples include, but are not limited to, taxol, cytochalasinB, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide,teniposide, vincristine, vinblastine, colchicine, doxorubicin,daunorubicin, dihydroxyanthracinedione, mitoxantrone, mithramycin,actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,tetracaine, lidocaine, propranolol, puromycin, maytansinoids, e.g.,maytansinol, rachelmycin (CC-1065), and analogs or homologs thereof.Radioactive ions include, but are not limited to iodine (e.g., iodine125 or iodine 131), strontium 89, phosphorous, palladium, cesium,iridium, phosphate, cobalt, yttrium 90, samarium 153, and praseodymium.Vaccines include compounds and preparations that are capable ofproviding immunity against one or more conditions related to infectiousdiseases such as influenza, measles, human papillomavirus (HPV), rabies,meningitis, whooping cough, tetanus, plague, hepatitis, and tuberculosisand can include mRNAs encoding infectious disease derived antigensand/or epitopes. Vaccines also include compounds and preparations thatdirect an immune response against cancer cells and can include mRNAsencoding tumor cell derived antigens, epitopes, and/or neoepitopes.Compounds eliciting immune responses may include vaccines,corticosteroids (e.g., dexamethasone), and other species. In someembodiments, a vaccine and/or a compound capable of eliciting an immuneresponse is administered intramuscularly via a composition including acompound according to Formula (I), (IA), (II), (IIa), (IIb), (IIc),(IId) or (IIe) (e.g., Compound 3, 18, 20, 25, 26, 29, 30, 60, 108-112,or 122). Other therapeutic and/or prophylactics include, but are notlimited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine,6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylatingagents (e.g., mechlorethamine, thiotepa chlorambucil, rachelmycin(CC-1065), melphalan, carmustine (BSNU), lomustine (CCNU),cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycinC, and cis-dichlorodiamine platinum (II) (DDP) cisplatin),anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine, vinblastine, taxol and maytansinoids).

In other embodiments, a therapeutic and/or prophylactic is a protein.Therapeutic proteins useful in the nanoparticles in the disclosureinclude, but are not limited to, gentamycin, amikacin, insulin,erythropoietin (EPO), granulocyte-colony stimulating factor (G-CSF),granulocyte-macrophage colony stimulating factor (GM-CSF), Factor VIR,luteinizing hormone-releasing hormone (LHRH) analogs, interferons,heparin, Hepatitis B surface antigen, typhoid vaccine, and choleravaccine.

Polynucleotides and Nucleic Acids

In some embodiments, a therapeutic agent is a polynucleotide or nucleicacid (e.g., ribonucleic acid or deoxyribonucleic acid). The term“polynucleotide,” in its broadest sense, includes any compound and/orsubstance that is or can be incorporated into an oligonucleotide chain.Exemplary polynucleotides for use in accordance with the presentdisclosure include, but are not limited to, one or more ofdeoxyribonucleic acid (DNA), ribonucleic acid (RNA) including messengermRNA (mRNA), hybrids thereof, RNAi-inducing agents, RNAi agents, siRNAs,shRNAs, miRNAs, antisense RNAs, ribozymes, catalytic DNA, RNAs thatinduce triple helix formation, aptamers, vectors, etc. In someembodiments, a therapeutic and/or prophylactic is an RNA. RNAs useful inthe compositions and methods described herein can be selected from thegroup consisting of, but are not limited to, shortmers, antagomirs,antisense, ribozymes, small interfering RNA (siRNA), asymmetricalinterfering RNA (aiRNA), microRNA (miRNA), Dicer-substrate RNA (dsRNA),small hairpin RNA (shRNA), transfer RNA (tRNA), messenger RNA (mRNA),and mixtures thereof. In certain embodiments, the RNA is an mRNA.

In certain embodiments, a therapeutic and/or prophylactic is an mRNA. AnmRNA may encode any polypeptide of interest, including any naturally ornon-naturally occurring or otherwise modified polypeptide. A polypeptideencoded by an mRNA may be of any size and may have any secondarystructure or activity. In some embodiments, a polypeptide encoded by anmRNA may have a therapeutic effect when expressed in a cell.

In other embodiments, a therapeutic and/or prophylactic is an siRNA. AnsiRNA may be capable of selectively knocking down or down regulatingexpression of a gene of interest. For example, an siRNA could beselected to silence a gene associated with a particular disease,disorder, or condition upon administration to a subject in need thereofof a nanoparticle composition including the siRNA. An siRNA may comprisea sequence that is complementary to an mRNA sequence that encodes a geneor protein of interest. In some embodiments, the siRNA may be animmunomodulatory siRNA.

In some embodiments, a therapeutic and/or prophylactic is an shRNA or avector or plasmid encoding the same. An shRNA may be produced inside atarget cell upon delivery of an appropriate construct to the nucleus.Constructs and mechanisms relating to shRNA are well known in therelevant arts.

Nucleic acids and polynucleotides useful in the disclosure typicallyinclude a first region of linked nucleosides encoding a polypeptide ofinterest (e.g., a coding region), a first flanking region located at the5′-terminus of the first region (e.g., a 5′-UTR), a second flankingregion located at the 3′-terminus of the first region (e.g., a 3′-UTR),at least one 5′-cap region, and a 3′-stabilizing region. In someembodiments, a nucleic acid or polynucleotide further includes a poly-Aregion or a Kozak sequence (e.g., in the 5′-UTR). In some cases,polynucleotides may contain one or more intronic nucleotide sequencescapable of being excised from the polynucleotide. In some embodiments, apolynucleotide or nucleic acid (e.g., an mRNA) may include a 5′ capstructure, a chain terminating nucleotide, a stem loop, a polyAsequence, and/or a polyadenylation signal. Any one of the regions of anucleic acid may include one or more alternative components (e.g., analternative nucleoside). For example, the 3′-stabilizing region maycontain an alternative nucleoside such as an L-nucleoside, an invertedthymidine, or a 2′-O-methyl nucleoside and/or the coding region, 5′-UTR,3′-UTR, or cap region may include an alternative nucleoside such as a5-substituted uridine (e.g., 5-methoxyuridine), a 1-substitutedpseudouridine (e.g., 1-methyl-pseudouridine or 1-ethyl-pseudouridine),and/or a 5-substituted cytidine (e.g., 5-methyl-cytidine).

Generally, the shortest length of a polynucleotide can be the length ofthe polynucleotide sequence that is sufficient to encode for adipeptide. In another embodiment, the length of the polynucleotidesequence is sufficient to encode for a tripeptide. In anotherembodiment, the length of the polynucleotide sequence is sufficient toencode for a tetrapeptide. In another embodiment, the length of thepolynucleotide sequence is sufficient to encode for a pentapeptide. Inanother embodiment, the length of the polynucleotide sequence issufficient to encode for a hexapeptide. In another embodiment, thelength of the polynucleotide sequence is sufficient to encode for aheptapeptide. In another embodiment, the length of the polynucleotidesequence is sufficient to encode for an octapeptide. In anotherembodiment, the length of the polynucleotide sequence is sufficient toencode for a nonapeptide. In another embodiment, the length of thepolynucleotide sequence is sufficient to encode for a decapeptide.

Examples of dipeptides that the alternative polynucleotide sequences canencode for include, but are not limited to, carnosine and anserine.

In some cases, a polynucleotide is greater than 30 nucleotides inlength. In another embodiment, the polynucleotide molecule is greaterthan 35 nucleotides in length. In another embodiment, the length is atleast 40 nucleotides. In another embodiment, the length is at least 45nucleotides. In another embodiment, the length is at least 55nucleotides. In another embodiment, the length is at least 50nucleotides. In another embodiment, the length is at least 60nucleotides. In another embodiment, the length is at least 80nucleotides. In another embodiment, the length is at least 90nucleotides. In another embodiment, the length is at least 100nucleotides. In another embodiment, the length is at least 120nucleotides. In another embodiment, the length is at least 140nucleotides. In another embodiment, the length is at least 160nucleotides. In another embodiment, the length is at least 180nucleotides. In another embodiment, the length is at least 200nucleotides. In another embodiment, the length is at least 250nucleotides. In another embodiment, the length is at least 300nucleotides. In another embodiment, the length is at least 350nucleotides. In another embodiment, the length is at least 400nucleotides. In another embodiment, the length is at least 450nucleotides. In another embodiment, the length is at least 500nucleotides. In another embodiment, the length is at least 600nucleotides. In another embodiment, the length is at least 700nucleotides. In another embodiment, the length is at least 800nucleotides. In another embodiment, the length is at least 900nucleotides. In another embodiment, the length is at least 1000nucleotides. In another embodiment, the length is at least 1100nucleotides. In another embodiment, the length is at least 1200nucleotides. In another embodiment, the length is at least 1300nucleotides. In another embodiment, the length is at least 1400nucleotides. In another embodiment, the length is at least 1500nucleotides. In another embodiment, the length is at least 1600nucleotides. In another embodiment, the length is at least 1800nucleotides. In another embodiment, the length is at least 2000nucleotides. In another embodiment, the length is at least 2500nucleotides. In another embodiment, the length is at least 3000nucleotides. In another embodiment, the length is at least 4000nucleotides. In another embodiment, the length is at least 5000nucleotides, or greater than 5000 nucleotides.

Nucleic acids and polynucleotides may include one or more naturallyoccurring components, including any of the canonical nucleotides A(adenosine), G (guanosine), C (cytosine), U (uridine), or T (thymidine).In one embodiment, all or substantially all of the nucleotidescomprising (a) the 5′-UTR, (b) the open reading frame (ORF), (c) the3′-UTR, (d) the poly A tail, and any combination of (a, b, c, or dabove) comprise naturally occurring canonical nucleotides A (adenosine),G (guanosine), C (cytosine), U (uridine), or T (thymidine).

Nucleic acids and polynucleotides may include one or more alternativecomponents, as described herein, which impart useful propertiesincluding increased stability and/or the lack of a substantial inductionof the innate immune response of a cell into which the polynucleotide isintroduced. For example, an alternative polynucleotide or nucleic acidexhibits reduced degradation in a cell into which the polynucleotide ornucleic acid is introduced, relative to a corresponding unalteredpolynucleotide or nucleic acid. These alternative species may enhancethe efficiency of protein production, intracellular retention of thepolynucleotides, and/or viability of contacted cells, as well as possessreduced immunogenicity.

Polynucleotides and nucleic acids may be naturally or non-naturallyoccurring. Polynucleotides and nucleic acids may include one or moremodified (e.g., altered or alternative) nucleobases, nucleosides,nucleotides, or combinations thereof. The nucleic acids andpolynucleotides useful in a nanoparticle composition can include anyuseful modification or alteration, such as to the nucleobase, the sugar,or the internucleoside linkage (e.g., to a linking phosphate/to aphosphodiester linkage/to the phosphodiester backbone). In certainembodiments, alterations (e.g., one or more alterations) are present ineach of the nucleobase, the sugar, and the internucleoside linkage.Alterations according to the present disclosure may be alterations ofribonucleic acids (RNAs) to deoxyribonucleic acids (DNAs), e.g., thesubstitution of the 2′-OH of the ribofuranosyl ring to 2′-H, threosenucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids(PNAs), locked nucleic acids (LNAs), or hybrids thereof. Additionalalterations are described herein.

Polynucleotides and nucleic acids may or may not be uniformly alteredalong the entire length of the molecule. For example, one or more or alltypes of nucleotide (e.g., purine or pyrimidine, or any one or more orall of A, G, U, C) may or may not be uniformly altered in apolynucleotide or nucleic acid, or in a given predetermined sequenceregion thereof. In some instances, all nucleotides X in a polynucleotide(or in a given sequence region thereof) are altered, wherein X may anyone of nucleotides A, G, U, C, or any one of the combinations A+G, A+U,A+C, G+U, G+C, U+C, A+G+U, A+G+C, G+U+C or A+G+C.

Different sugar alterations and/or internucleoside linkages (e.g.,backbone structures) may exist at various positions in a polynucleotide.One of ordinary skill in the art will appreciate that the nucleotideanalogs or other alteration(s) may be located at any position(s) of apolynucleotide such that the function of the polynucleotide is notsubstantially decreased. An alteration may also be a 5′- or 3′-terminalalteration. In some embodiments, the polynucleotide includes analteration at the 3′-terminus. The polynucleotide may contain from about1% to about 100% alternative nucleotides (either in relation to overallnucleotide content, or in relation to one or more types of nucleotide,i.e., any one or more of A, G, U or C) or any intervening percentage(e.g., from 1% to 20%, from 1% to 25%, from 1% to 50%, from 1% to 60%,from 1% to 70%, from 1% to 80%, from 1% to 90%, from 1% to 95%, from 10%to 20%, from 10% to 25%, from 10% to 50%, from 10% to 60%, from 10% to70%, from 10% to 80%, from 10% to 90%, from 10% to 95%, from 10% to100%, from 20% to 25%, from 20% to 50%, from 20% to 60%, from 20% to70%, from 20% to 80%, from 20% to 90%, from 20% to 95%, from 20% to100%, from 50% to 60%, from 50% to 70%, from 50% to 80%, from 50% to90%, from 50% to 95%, from 50% to 100%, from 70% to 80%, from 70% to90%, from 70% to 95%, from 70% to 100%, from 80% to 90%, from 80% to95%, from 80% to 100%, from 90% to 95%, from 90% to 100%, and from 95%to 100%). It will be understood that any remaining percentage isaccounted for by the presence of a canonical nucleotide (e.g., A, G, U,or C).

Polynucleotides may contain at a minimum zero and at maximum 100%alternative nucleotides, or any intervening percentage, such as at least5% alternative nucleotides, at least 10% alternative nucleotides, atleast 25% alternative nucleotides, at least 50% alternative nucleotides,at least 80% alternative nucleotides, or at least 90% alternativenucleotides. For example, polynucleotides may contain an alternativepyrimidine such as an alternative uracil or cytosine. In someembodiments, at least 5%, at least 10%, at least 25%, at least 50%, atleast 80%, at least 90% or 100% of the uracil in a polynucleotide isreplaced with an alternative uracil (e.g., a 5-substituted uracil). Thealternative uracil can be replaced by a compound having a single uniquestructure, or can be replaced by a plurality of compounds havingdifferent structures (e.g., 2, 3, 4 or more unique structures). In someinstances, at least 5%, at least 10%, at least 25%, at least 50%, atleast 80%, at least 90% or 100% of the cytosine in the polynucleotide isreplaced with an alternative cytosine (e.g., a 5-substituted cytosine).The alternative cytosine can be replaced by a compound having a singleunique structure, or can be replaced by a plurality of compounds havingdifferent structures (e.g., 2, 3, 4 or more unique structures).

In some instances, nucleic acids do not substantially induce an innateimmune response of a cell into which the polynucleotide (e.g., mRNA) isintroduced. Features of an induced innate immune response include 1)increased expression of pro-inflammatory cytokines, 2) activation ofintracellular PRRs (RIG-I, MDA5, etc., and/or 3) termination orreduction in protein translation.

The nucleic acids can optionally include other agents (e.g.,RNAi-inducing agents, RNAi agents, siRNAs, shRNAs, miRNAs, antisenseRNAs, ribozymes, catalytic DNA, tRNA, RNAs that induce triple helixformation, aptamers, vectors). In some embodiments, the nucleic acidsmay include one or more messenger RNAs (mRNAs) having one or morealternative nucleoside or nucleotides (i.e., alternative mRNAmolecules).

In some embodiments, a nucleic acid (e.g. mRNA) molecule, formula,composition or method associated therewith comprises one or morepolynucleotides comprising features as described in WO2002/098443,WO2003/051401, WO2008/052770, WO2009127230, WO2006122828, WO2008/083949,WO2010088927, WO2010/037539, WO2004/004743, WO2005/016376,WO2006/024518, WO2007/095976, WO2008/014979, WO2008/077592,WO2009/030481, WO2009/095226, WO2011069586, WO2011026641, WO2011/144358,WO2012019780, WO2012013326, WO2012089338, WO2012113513, WO2012116811,WO2012116810, WO2013113502, WO2013113501, WO2013113736, WO2013143698,WO2013143699, WO2013143700, WO2013/120626, WO2013120627, WO2013120628,WO2013120629, WO2013174409, WO2014127917, WO2015/024669, WO2015/024668,WO2015/024667, WO2015/024665, WO2015/024666, WO2015/024664,WO2015101415, WO2015101414, WO2015024667, WO2015062738, WO2015101416,all of which are incorporated by reference herein.

Nucleobase Alternatives

The alternative nucleosides and nucleotides can include an alternativenucleobase. A nucleobase of a nucleic acid is an organic base such as apurine or pyrimidine or a derivative thereof. A nucleobase may be acanonical base (e.g., adenine, guanine, uracil, thymine, and cytosine).These nucleobases can be altered or wholly replaced to providepolynucleotide molecules having enhanced properties, e.g., increasedstability such as resistance to nucleases. Non-canonical or modifiedbases may include, for example, one or more substitutions ormodifications including but not limited to alkyl, aryl, halo, oxo,hydroxyl, alkyloxy, and/or thio substitutions; one or more fused or openrings; oxidation; and/or reduction.

Alternative nucleotide base pairing encompasses not only the standardadenine-thymine, adenine-uracil, or guanine-cytosine base pairs, butalso base pairs formed between nucleotides and/or alternativenucleotides including non-standard or alternative bases, wherein thearrangement of hydrogen bond donors and hydrogen bond acceptors permitshydrogen bonding between a non-standard base and a standard base orbetween two complementary non-standard base structures. One example ofsuch non-standard base pairing is the base pairing between thealternative nucleotide inosine and adenine, cytosine, or uracil.

In some embodiments, the nucleobase is an alternative uracil. Exemplarynucleobases and nucleosides having an alternative uracil includepseudouridine (ψ), pyridin-4-one ribonucleoside, 5-aza-uracil,6-aza-uracil, 2-thio-5-aza-uracil, 2-thio-uracil (s²U), 4-thio-uracil(s⁴U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uracil(ho⁵U), 5-aminoallyl-uracil, 5-halo-uracil (e.g., 5-iodo-uracil or5-bromo-uracil), 3-methyl-uracil (m³U), 5-methoxy-uracil (mo⁵U), uracil5-oxyacetic acid (cmo⁵U), uracil 5-oxyacetic acid methyl ester (mcmo⁵U),5-carboxymethyl-uracil (cm⁵U), 1-carboxymethyl-pseudouridine,5-carboxyhydroxymethyl-uracil (chm⁵U), 5-carboxyhydroxymethyl-uracilmethyl ester (mchm⁵U), 5-methoxycarbonylmethyl-uracil (mcm⁵U),5-methoxycarbonylmethyl-2-thio-uracil (mcm⁵s²U),5-aminomethyl-2-thio-uracil (nm⁵ s²U), 5-methylaminomethyl-uracil(mnm⁵U), 5-methylaminomethyl-2-thio-uracil (mnm⁵ s²U),5-methylaminomethyl-2-seleno-uracil (mnm⁵se²U), 5-carbamoylmethyl-uracil(ncm⁵U), 5-carboxymethylaminomethyl-uracil (cmnm⁵U),5-carboxymethylaminomethyl-2-thio-uracil (cmnm⁵ s²U), 5-propynyl-uracil,1-propynyl-pseudouracil, 5-taurinomethyl-uracil (τm⁵U),1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uracil (m⁵ s²U),1-taurinomethyl-4-thio-pseudouridine, 5-methyl-uracil (m⁵U, i.e., havingthe nucleobase deoxythymine), 1-methyl-pseudouridine (m¹ψ),1-ethyl-pseudouridine (Et¹ψ), 5-methyl-2-thio-uracil (m⁵s²U),1-methyl-4-thio-pseudouridine (m¹s⁴ψ), 4-thio-1-methyl-pseudouridine,3-methyl-pseudouridine (m³ψ), 2-thio-1-methyl-pseudouridine,1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine,dihydrouracil (D), dihydropseudouridine, 5,6-dihydrouracil,5-methyl-dihydrouracil (m⁵D), 2-thio-dihydrouracil,2-thio-dihydropseudouridine, 2-methoxy-uracil, 2-methoxy-4-thio-uracil,4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine,N1-methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uracil (acp³U),1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp³ψ),5-(isopentenylaminomethyl)uracil (inm⁵U),5-(isopentenylaminomethyl)-2-thio-uracil (inm⁵ s²U),5,2′-O-dimethyl-uridine (m⁵Um), 2-thio-2′-O_methyl-uridine (s²Um),5-methoxycarbonylmethyl-2′-O-methyl-uridine (mcm⁵Um),5-carbamoylmethyl-2′-O-methyl-uridine (ncm⁵Um),5-carboxymethylaminomethyl-2′-O-methyl-uridine (cmnm⁵Um),3,2′-O-dimethyl-uridine (m³Um), and5-(isopentenylaminomethyl)-2′-O-methyl-uridine (inm⁵Um), 1-thio-uracil,deoxythymidine, 5-(2-carbomethoxyvinyl)-uracil,5-(carbamoylhydroxymethyl)-uracil, 5-carbamoylmethyl-2-thio-uracil,5-carboxymethyl-2-thio-uracil, 5-cyanomethyl-uracil,5-methoxy-2-thio-uracil, and 5-[3-(1-E-propenylamino)]uracil.

In some embodiments, the nucleobase is an alternative cytosine.Exemplary nucleobases and nucleosides having an alternative cytosineinclude 5-aza-cytosine, 6-aza-cytosine, pseudoisocytidine,3-methyl-cytosine (m3C), N4-acetyl-cytosine (ac4C), 5-formyl-cytosine(f5C), N4-methyl-cytosine (m4C), 5-methyl-cytosine (m5C),5-halo-cytosine (e.g., 5-iodo-cytosine), 5-hydroxymethyl-cytosine(hm5C), 1-methyl-pseudoisocytidine, pyrrolo-cytosine,pyrrolo-pseudoisocytidine, 2-thio-cytosine (s2C),2-thio-5-methyl-cytosine, 4-thio-pseudoisocytidine,4-thio-1-methyl-pseudoisocytidine,4-thio-1-methyl-1-deaza-pseudoisocytidine,1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine,5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine,2-methoxy-cytosine, 2-methoxy-5-methyl-cytosine,4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine,lysidine (k2C), 5,2′-O-dimethyl-cytidine (m5Cm),N4-acetyl-2′-O-methyl-cytidine (ac4Cm), N4,2′-O-dimethyl-cytidine(m4Cm), 5-formyl-2′-O-methyl-cytidine (f5Cm),N4,N4,2′-O-trimethyl-cytidine (m42Cm), 1-thio-cytosine,5-hydroxy-cytosine, 5-(3-azidopropyl)-cytosine, and5-(2-azidoethyl)-cytosine.

In some embodiments, the nucleobase is an alternative adenine. Exemplarynucleobases and nucleosides having an alternative adenine include2-amino-purine, 2,6-diaminopurine, 2-amino-6-halo-purine (e.g.,2-amino-6-chloro-purine), 6-halo-purine (e.g., 6-chloro-purine),2-amino-6-methyl-purine, 8-azido-adenine, 7-deaza-adenine,7-deaza-8-aza-adenine, 7-deaza-2-amino-purine,7-deaza-8-aza-2-amino-purine, 7-deaza-2,6-diaminopurine,7-deaza-8-aza-2,6-diaminopurine, 1-methyl-adenine (m1A),2-methyl-adenine (m2A), N6-methyl-adenine (m6A),2-methylthio-N6-methyl-adenine (ms2m6A), N6-isopentenyl-adenine (i6A),2-methylthio-N6-isopentenyl-adenine (ms2i6A),N6-(cis-hydroxyisopentenyl)adenine (io6A),2-methylthio-N6-(cis-hydroxyisopentenyl)adenine (ms2io6A),N6-glycinylcarbamoyl-adenine (g6A), N6-threonylcarbamoyl-adenine (t6A),N6-methyl-N6-threonylcarbamoyl-adenine (m6t6A),2-methylthio-N6-threonylcarbamoyl-adenine (ms2g6A),N6,N6-dimethyl-adenine (m62A), N6-hydroxynorvalylcarbamoyl-adenine(hn6A), 2-methylthio-N6-hydroxynorvalylcarbamoyl-adenine (ms2hn6A),N6-acetyl-adenine (ac6A), 7-methyl-adenine, 2-methylthio-adenine,2-methoxy-adenine, N6,2′-O-dimethyl-adenosine (m6Am),N6,N6,2′-O-trimethyl-adenosine (m62Am), 1,2′-O-dimethyl-adenosine(m1Am), 2-amino-N6-methyl-purine, 1-thio-adenine, 8-azido-adenine,N6-(19-amino-pentaoxanonadecyl)-adenine, 2,8-dimethyl-adenine,N6-formyl-adenine, and N6-hydroxymethyl-adenine.

In some embodiments, the nucleobase is an alternative guanine. Exemplarynucleobases and nucleosides having an alternative guanine includeinosine (I), 1-methyl-inosine (m1I), wyosine (imG), methylwyosine(mimG), 4-demethyl-wyosine (imG-14), isowyosine (imG2), wybutosine (yW),peroxywybutosine (o2yW), hydroxywybutosine (OHyW), undermodifiedhydroxywybutosine (OHyW*), 7-deaza-guanine, queuosine (Q),epoxyqueuosine (oQ), galactosyl-queuosine (galQ), mannosyl-queuosine(manQ), 7-cyano-7-deaza-guanine (preQ0), 7-aminomethyl-7-deaza-guanine(preQ1), archaeosine (G+), 7-deaza-8-aza-guanine, 6-thio-guanine,6-thio-7-deaza-guanine, 6-thio-7-deaza-8-aza-guanine, 7-methyl-guanine(m7G), 6-thio-7-methyl-guanine, 7-methyl-inosine, 6-methoxy-guanine,1-methyl-guanine (m1G), N2-methyl-guanine (m2G), N2,N2-dimethyl-guanine(m22G), N2,7-dimethyl-guanine (m2,7G), N2, N2,7-dimethyl-guanine(m2,2,7G), 8-oxo-guanine, 7-methyl-8-oxo-guanine,1-methyl-6-thio-guanine, N2-methyl-6-thio-guanine,N2,N2-dimethyl-6-thio-guanine, N2-methyl-2′-O-methyl-guanosine (m2Gm),N2,N2-dimethyl-2′-O-methyl-guanosine (m22Gm),1-methyl-2′-O-methyl-guanosine (m1Gm),N2,7-dimethyl-2′-O-methyl-guanosine (m2,7Gm), 2′-O-methyl-inosine (Im),1,2′-O-dimethyl-inosine (m1Im), 1-thio-guanine, and O-6-methyl-guanine.

The alternative nucleobase of a nucleotide can be independently apurine, a pyrimidine, a purine or pyrimidine analog. For example, thenucleobase can be an alternative to adenine, cytosine, guanine, uracil,or hypoxanthine. In another embodiment, the nucleobase can also include,for example, naturally-occurring and synthetic derivatives of a base,including pyrazolo[3,4-d]pyrimidines, 5-methylcytosine (5-me-C),5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine,6-methyl and other alkyl derivatives of adenine and guanine, 2-propyland other alkyl derivatives of adenine and guanine, 2-thiouracil,2-thiothymine and 2-thiocytosine, 5-propynyl uracil and cytosine, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil,8-halo (e.g., 8-bromo), 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxy andother 8-substituted adenines and guanines, 5-halo particularly 5-bromo,5-trifluoromethyl and other 5-substituted uracils and cytosines,7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine,deazaguanine, 7-deazaguanine, 3-deazaguanine, deazaadenine,7-deazaadenine, 3-deazaadenine, pyrazolo[3,4-d]pyrimidine,imidazo[1,5-a]1,3,5 triazinones, 9-deazapurines,imidazo[4,5-d]pyrazines, thiazolo[4,5-d]pyrimidines, pyrazin-2-ones,1,2,4-triazine, pyridazine; or 1,3,5 triazine. When the nucleotides aredepicted using the shorthand A, G, C, T or U, each letter refers to therepresentative base and/or derivatives thereof, e.g., A includes adenineor adenine analogs, e.g., 7-deaza adenine).

Alterations on the Sugar

Nucleosides include a sugar molecule (e.g., a 5-carbon or 6-carbonsugar, such as pentose, ribose, arabinose, xylose, glucose, galactose,or a deoxy derivative thereof) in combination with a nucleobase, whilenucleotides are nucleosides containing a nucleoside and a phosphategroup or alternative group (e.g., boranophosphate, thiophosphate,selenophosphate, phosphonate, alkyl group, amidate, and glycerol). Anucleoside or nucleotide may be a canonical species, e.g., a nucleosideor nucleotide including a canonical nucleobase, sugar, and, in the caseof nucleotides, a phosphate group, or may be an alternative nucleosideor nucleotide including one or more alternative components. For example,alternative nucleosides and nucleotides can be altered on the sugar ofthe nucleoside or nucleotide. In some embodiments, the alternativenucleosides or nucleotides include the structure:

In each of the Formulae IV, V, VI and VII,

each of m and n is independently, an integer from 0 to 5,

each of U and U′ independently, is O, S, N(R^(U))_(nu), orC(R^(U))_(nu), wherein nu is an integer from 0 to 2 and each R^(U) is,independently, H, halo, or optionally substituted alkyl;

each of R^(1′), R^(2′), R^(1″), R^(2″), R¹, R², R³, R⁴, and R⁵ is,independently, if present, H, halo, hydroxy, thiol, optionallysubstituted alkyl, optionally substituted alkoxy, optionally substitutedalkenyloxy, optionally substituted alkynyloxy, optionally substitutedaminoalkoxy, optionally substituted alkoxyalkoxy, optionally substitutedhydroxyalkoxy, optionally substituted amino, azido, optionallysubstituted aryl, optionally substituted aminoalkyl, optionallysubstituted aminoalkenyl, optionally substituted aminoalkynyl, orabsent; wherein the combination of R³ with one or more of R^(1′),R^(1″), R^(2′), R^(2″), or R⁵ (e.g., the combination of R^(1′) and R³,the combination of R^(1′) and R³, the combination of R^(2′) and R³, thecombination of R^(2″) and R³, or the combination of R⁵ and R³) can jointogether to form optionally substituted alkylene or optionallysubstituted heteroalkylene and, taken together with the carbons to whichthey are attached, provide an optionally substituted heterocyclyl (e.g.,a bicyclic, tricyclic, or tetracyclic heterocyclyl); wherein thecombination of R⁵ with one or more of R^(1′), R¹″, R^(2′), or R²″ (e.g.,the combination of R^(1′) and R⁵, the combination of R^(1′) and R⁵, thecombination of R^(2′) and R⁵, or the combination of R^(2″) and R⁵) canjoin together to form optionally substituted alkylene or optionallysubstituted heteroalkylene and, taken together with the carbons to whichthey are attached, provide an optionally substituted heterocyclyl (e.g.,a bicyclic, tricyclic, or tetracyclic heterocyclyl); and wherein thecombination of R⁴ and one or more of R^(1′), R^(1′), R^(2′), R^(2″), R³,or R⁵ can join together to form optionally substituted alkylene oroptionally substituted heteroalkylene and, taken together with thecarbons to which they are attached, provide an optionally substitutedheterocyclyl (e.g., a bicyclic, tricyclic, or tetracyclic heterocyclyl);each of m′ and m″ is, independently, an integer from 0 to 3 (e.g., from0 to 2, from 0 to 1, from 1 to 3, or from 1 to 2);

each of Y¹, Y², and Y³, is, independently, O, S, Se, —NR^(N1)—,optionally substituted alkylene, or optionally substitutedheteroalkylene, wherein R^(N1) is H, optionally substituted alkyl,optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted aryl, or absent; each Y⁴ is, independently, H,hydroxy, thiol, boranyl, optionally substituted alkyl, optionallysubstituted alkenyl, optionally substituted alkynyl, optionallysubstituted alkoxy, optionally substituted alkenyloxy, optionallysubstituted alkynyloxy, optionally substituted thioalkoxy, optionallysubstituted alkoxyalkoxy, or optionally substituted amino;

each Y⁵ is, independently, O, S, Se, optionally substituted alkylene(e.g., methylene), or optionally substituted heteroalkylene; and

B is a nucleobase, either modified or unmodified. In some embodiments,the 2′-hydroxy group (OH) can be modified or replaced with a number ofdifferent substituents. Exemplary substitutions at the 2′-positioninclude, but are not limited to, H, azido, halo (e.g., fluoro),optionally substituted C₁₋₆ alkyl (e.g., methyl); optionally substitutedC₁₋₆ alkoxy (e.g., methoxy or ethoxy); optionally substituted C₆₋₁₀aryloxy; optionally substituted C₃₋₈ cycloalkyl; optionally substitutedC₆₋₁₀ aryl-C₁₋₆ alkoxy, optionally substituted C₁₋₁₂ (heterocyclyl)oxy;a sugar (e.g., ribose, pentose, or any described herein); apolyethyleneglycol (PEG), —O(CH₂CH₂O)_(n)CH₂CH₂OR, where R is H oroptionally substituted alkyl, and n is an integer from 0 to 20 (e.g.,from 0 to 4, from 0 to 8, from 0 to 10, from 0 to 16, from 1 to 4, from1 to 8, from 1 to 10, from 1 to 16, from 1 to 20, from 2 to 4, from 2 to8, from 2 to 10, from 2 to 16, from 2 to 20, from 4 to 8, from 4 to 10,from 4 to 16, and from 4 to 20); “locked” nucleic acids (LNA) in whichthe 2′-hydroxy is connected by a C₁₋₆ alkylene or C₁₋₆ heteroalkylenebridge to the 4′-carbon of the same ribose sugar, where exemplarybridges included methylene, propylene, ether, or amino bridges;aminoalkyl, as defined herein; aminoalkoxy, as defined herein; amino asdefined herein; and amino acid, as defined herein.

Generally, RNA includes the sugar group ribose, which is a 5-memberedring having an oxygen. Exemplary, non-limiting alternative nucleotidesinclude replacement of the oxygen in ribose (e.g., with S, Se, oralkylene, such as methylene or ethylene); addition of a double bond(e.g., to replace ribose with cyclopentenyl or cyclohexenyl); ringcontraction of ribose (e.g., to form a 4-membered ring of cyclobutane oroxetane); ring expansion of ribose (e.g., to form a 6- or 7-memberedring having an additional carbon or heteroatom, such as foranhydrohexitol, altritol, mannitol, cyclohexanyl, cyclohexenyl, andmorpholino (that also has a phosphoramidate backbone)); multicyclicforms (e.g., tricyclo and “unlocked” forms, such as glycol nucleic acid(GNA) (e.g., R-GNA or S-GNA, where ribose is replaced by glycol unitsattached to phosphodiester bonds), threose nucleic acid (TNA, whereribose is replace with α-L-threofuranosyl-(3′→2′)), and peptide nucleicacid (PNA, where 2-amino-ethyl-glycine linkages replace the ribose andphosphodiester backbone).

In some embodiments, the sugar group contains one or more carbons thatpossess the opposite stereochemical configuration of the correspondingcarbon in ribose. Thus, a polynucleotide molecule can includenucleotides containing, e.g., arabinose or L-ribose, as the sugar.

In some embodiments, the polynucleotide includes at least one nucleosidewherein the sugar is L-ribose, 2′-O-methyl-ribose, 2′-fluoro-ribose,arabinose, hexitol, an LNA, or a PNA.

Alterations on the Internucleoside Linkage

Alternative nucleotides can be altered on the internucleoside linkage(e.g., phosphate backbone). Herein, in the context of the polynucleotidebackbone, the phrases “phosphate” and “phosphodiester” are usedinterchangeably. Backbone phosphate groups can be altered by replacingone or more of the oxygen atoms with a different substituent.

The alternative nucleotides can include the wholesale replacement of anunaltered phosphate moiety with another internucleoside linkage asdescribed herein. Examples of alternative phosphate groups include, butare not limited to, phosphorothioate, phosphoroselenates,boranophosphates, boranophosphate esters, hydrogen phosphonates,phosphoramidates, phosphorodiamidates, alkyl or aryl phosphonates, andphosphotriesters. Phosphorodithioates have both non-linking oxygensreplaced by sulfur. The phosphate linker can also be altered by thereplacement of a linking oxygen with nitrogen (bridgedphosphoramidates), sulfur (bridged phosphorothioates), and carbon(bridged methylene-phosphonates).

The alternative nucleosides and nucleotides can include the replacementof one or more of the non-bridging oxygens with a borane moiety (BH₃),sulfur (thio), methyl, ethyl, and/or methoxy. As a non-limiting example,two non-bridging oxygens at the same position (e.g., the alpha (α), beta(β) or gamma (γ) position) can be replaced with a sulfur (thio) and amethoxy.

The replacement of one or more of the oxygen atoms at the a position ofthe phosphate moiety (e.g., α-thio phosphate) is provided to conferstability (such as against exonucleases and endonucleases) to RNA andDNA through the unnatural phosphorothioate backbone linkages.Phosphorothioate DNA and RNA have increased nuclease resistance andsubsequently a longer half-life in a cellular environment.

Other internucleoside linkages that may be employed according to thepresent disclosure, including internucleoside linkages which do notcontain a phosphorous atom, are described herein.

Internal Ribosome Entry Sites

Polynucleotides may contain an internal ribosome entry site (IRES). AnIRES may act as the sole ribosome binding site, or may serve as one ofmultiple ribosome binding sites of an mRNA. A polynucleotide containingmore than one functional ribosome binding site may encode severalpeptides or polypeptides that are translated independently by theribosomes (e.g., multicistronic mRNA). When polynucleotides are providedwith an IRES, further optionally provided is a second translatableregion. Examples of IRES sequences that can be used according to thepresent disclosure include without limitation, those from picornaviruses(e.g., FMDV), pest viruses (CFFV), polio viruses (PV),encephalomyocarditis viruses (ECMV), foot-and-mouth disease viruses(FMDV), hepatitis C viruses (HCV), classical swine fever viruses (CSFV),murine leukemia virus (MLV), simian immune deficiency viruses (SIV) orcricket paralysis viruses (CrPV).

5′-Cap Structure

A polynucleotide (e.g., an mRNA) may include a 5′-cap structure. The5′-cap structure of a polynucleotide is involved in nuclear export andincreasing polynucleotide stability and binds the mRNA Cap BindingProtein (CBP), which is responsible for polynucleotide stability in thecell and translation competency through the association of CBP withpoly-A binding protein to form the mature cyclic mRNA species. The capfurther assists the removal of 5′-proximal introns removal during mRNAsplicing.

Endogenous polynucleotide molecules may be 5′-end capped generating a5′-ppp-5′-triphosphate linkage between a terminal guanosine cap residueand the 5′-terminal transcribed sense nucleotide of the polynucleotide.This 5′-guanylate cap may then be methylated to generate anN7-methyl-guanylate residue. The ribose sugars of the terminal and/oranteterminal transcribed nucleotides of the 5′ end of the polynucleotidemay optionally also be 2′-O-methylated. 5′-decapping through hydrolysisand cleavage of the guanylate cap structure may target a polynucleotidemolecule, such as an mRNA molecule, for degradation.

Alterations to polynucleotides may generate a non-hydrolyzable capstructure preventing decapping and thus increasing polynucleotidehalf-life. Because cap structure hydrolysis requires cleavage of5′-ppp-5′ phosphorodiester linkages, alternative nucleotides may be usedduring the capping reaction. For example, a Vaccinia Capping Enzyme fromNew England Biolabs (Ipswich, Mass.) may be used with α-thio-guanosinenucleotides according to the manufacturer's instructions to create aphosphorothioate linkage in the 5′-ppp-5′ cap. Additional alternativeguanosine nucleotides may be used such as α-methyl-phosphonate andseleno-phosphate nucleotides.

Additional alterations include, but are not limited to, 2′-O-methylationof the ribose sugars of 5′-terminal and/or 5′-anteterminal nucleotidesof the polynucleotide (as mentioned above) on the 2′-hydroxy group ofthe sugar. Multiple distinct 5′-cap structures can be used to generatethe 5′-cap of a polynucleotide, such as an mRNA molecule.

5′-Cap structures include those described in International PatentPublication Nos. WO2008127688, WO 2008016473, and WO 2011015347, the capstructures of each of which are incorporated herein by reference.

Cap analogs, which herein are also referred to as synthetic cap analogs,chemical caps, chemical cap analogs, or structural or functional capanalogs, differ from natural (i.e., endogenous, wild-type, orphysiological) 5′-caps in their chemical structure, while retaining capfunction. Cap analogs may be chemically (i.e., non-enzymatically) orenzymatically synthesized and/linked to a polynucleotide.

For example, the Anti-Reverse Cap Analog (ARCA) cap contains twoguanosines linked by a 5′-5′-triphosphate group, wherein one guanosinecontains an N7-methyl group as well as a 3′-O-methyl group (i.e.,N7,3′-O-dimethyl-guanosine-5′-triphosphate-5′-guanosine, m⁷G-3′mppp-G,which may equivalently be designated 3′ O-Me-m7G(5′)ppp(5′)G). The 3′-Oatom of the other, unaltered, guanosine becomes linked to the5′-terminal nucleotide of the capped polynucleotide (e.g., an mRNA). TheN7- and 3′-O-methlyated guanosine provides the terminal moiety of thecapped polynucleotide (e.g., mRNA).

Another exemplary cap is mCAP, which is similar to ARCA but has a2′-O-methyl group on guanosine (i.e.,N7,2′-O-dimethyl-guanosine-5′-triphosphate-5′-guanosine, m⁷Gm-ppp-G).

A cap may be a dinucleotide cap analog. As a non-limiting example, thedinucleotide cap analog may be modified at different phosphate positionswith a boranophosphate group or a phophoroselenoate group such as thedinucleotide cap analogs described in U.S. Pat. No. 8,519,110, the capstructures of which are herein incorporated by reference.

Alternatively, a cap analog may be a N7-(4-chlorophenoxyethyl)substituted dinucleotide cap analog known in the art and/or describedherein. Non-limiting examples of N7-(4-chlorophenoxyethyl) substituteddinucleotide cap analogs include aN7-(4-chlorophenoxyethyl)-G(5′)ppp(5′)G and aN7-(4-chlorophenoxyethyl)-m3′-OG(5′)ppp(5′)G cap analog (see, e.g., thevarious cap analogs and the methods of synthesizing cap analogsdescribed in Kore et al. Bioorganic & Medicinal Chemistry 201321:4570-4574; the cap structures of which are herein incorporated byreference). In other instances, a cap analog useful in thepolynucleotides of the present disclosure is a4-chloro/bromophenoxyethyl analog.

While cap analogs allow for the concomitant capping of a polynucleotidein an in vitro transcription reaction, up to 20% of transcripts remainuncapped. This, as well as the structural differences of a cap analogfrom endogenous 5′-cap structures of polynucleotides produced by theendogenous, cellular transcription machinery, may lead to reducedtranslational competency and reduced cellular stability.

Alternative polynucleotides may also be capped post-transcriptionally,using enzymes, in order to generate more authentic 5′-cap structures. Asused herein, the phrase “more authentic” refers to a feature thatclosely mirrors or mimics, either structurally or functionally, anendogenous or wild type feature. That is, a “more authentic” feature isbetter representative of an endogenous, wild-type, natural orphysiological cellular function, and/or structure as compared tosynthetic features or analogs of the prior art, or which outperforms thecorresponding endogenous, wild-type, natural, or physiological featurein one or more respects. Non-limiting examples of more authentic 5′-capstructures useful in the polynucleotides of the present disclosure arethose which, among other things, have enhanced binding of cap bindingproteins, increased half life, reduced susceptibility to5′-endonucleases, and/or reduced 5′-decapping, as compared to synthetic5′-cap structures known in the art (or to a wild-type, natural orphysiological 5′-cap structure). For example, recombinant Vaccinia VirusCapping Enzyme and recombinant 2′-O-methyltransferase enzyme can createa canonical 5′-5′-triphosphate linkage between the 5′-terminalnucleotide of a polynucleotide and a guanosine cap nucleotide whereinthe cap guanosine contains an N7-methylation and the 5′-terminalnucleotide of the polynucleotide contains a 2′-O-methyl. Such astructure is termed the Cap1 structure. This cap results in a highertranslational-competency, cellular stability, and a reduced activationof cellular pro-inflammatory cytokines, as compared, e.g., to other5′cap analog structures known in the art. Other exemplary cap structuresinclude 7mG(5′)ppp(5′)N, pN2p (Cap 0), 7mG(5′)ppp(5′)N1mpNp (Cap 1),7mG(5′)-ppp(5′)N1mpN2mp (Cap 2), andm(7)Gpppm(3)(6,6,2′)Apm(2′)Apm(2′)Cpm(2)(3,2′)Up (Cap 4).

Because the alternative polynucleotides may be cappedpost-transcriptionally, and because this process is more efficient,nearly 100% of the alternative polynucleotides may be capped. This is incontrast to ˜80% when a cap analog is linked to an polynucleotide in thecourse of an in vitro transcription reaction.

5′-terminal caps may include endogenous caps or cap analogs. A5′-terminal cap may include a guanosine analog. Useful guanosine analogsinclude inosine, N1-methyl-guanosine, 2′-fluoro-guanosine,7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine,and 2-azido-guanosine.

In some cases, a polynucleotide contains a modified 5′-cap. Amodification on the 5′-cap may increase the stability of polynucleotide,increase the half-life of the polynucleotide, and could increase thepolynucleotide translational efficiency. The modified 5′-cap mayinclude, but is not limited to, one or more of the followingmodifications: modification at the 2′- and/or 3′-position of a cappedguanosine triphosphate (GTP), a replacement of the sugar ring oxygen(that produced the carbocyclic ring) with a methylene moiety (CH₂), amodification at the triphosphate bridge moiety of the cap structure, ora modification at the nucleobase (G) moiety.

5′-UTRs

A 5′-UTR may be provided as a flanking region to polynucleotides (e.g.,mRNAs). A 5′-UTR may be homologous or heterologous to the coding regionfound in a polynucleotide. Multiple 5′-UTRs may be included in theflanking region and may be the same or of different sequences. Anyportion of the flanking regions, including none, may be codon optimizedand any may independently contain one or more different structural orchemical alterations, before and/or after codon optimization.

Shown in Table 21 in U.S. Provisional Application No. 61/775,509, and inTable 21 and in Table 22 in U.S. Provisional Application No. 61/829,372,of which are incorporated herein by reference, is a listing of the startand stop site of alternative polynucleotides (e.g., mRNA). In Table 21each 5′-UTR (5′-UTR-005 to 5′-UTR 68511) is identified by its start andstop site relative to its native or wild type (homologous) transcript(ENST; the identifier used in the ENSEMBL database).

To alter one or more properties of a polynucleotide (e.g., mRNA),5′-UTRs which are heterologous to the coding region of an alternativepolynucleotide (e.g., mRNA) may be engineered. The polynucleotides(e.g., mRNA) may then be administered to cells, tissue or organisms andoutcomes such as protein level, localization, and/or half-life may bemeasured to evaluate the beneficial effects the heterologous 5′-UTR mayhave on the alternative polynucleotides (mRNA). Variants of the 5′-UTRsmay be utilized wherein one or more nucleotides are added or removed tothe termini, including A, T, C or G. 5′-UTRs may also becodon-optimized, or altered in any manner described herein.

5′-UTRs, 3′-UTRs, and Translation Enhancer Elements (TEEs)

The 5′-UTR of a polynucleotides (e.g., mRNA) may include at least onetranslation enhancer element. The term “translational enhancer element”refers to sequences that increase the amount of polypeptide or proteinproduced from a polynucleotide. As a non-limiting example, the TEE maybe located between the transcription promoter and the start codon. Thepolynucleotides (e.g., mRNA) with at least one TEE in the 5′-UTR mayinclude a cap at the 5′-UTR. Further, at least one TEE may be located inthe 5′-UTR of polynucleotides (e.g., mRNA) undergoing cap-dependent orcap-independent translation.

In one aspect, TEEs are conserved elements in the UTR which can promotetranslational activity of a polynucleotide such as, but not limited to,cap-dependent or cap-independent translation. The conservation of thesesequences has been previously shown by Panek et al. (Nucleic AcidsResearch, 2013, 1-10) across 14 species including humans.

In one non-limiting example, the TEEs known may be in the 5′-leader ofthe Gtx homeodomain protein (Chappell et al., Proc. Natl. Acad. Sci. USA101:9590-9594, 2004, the TEEs of which are incorporated herein byreference).

In another non-limiting example, TEEs are disclosed as SEQ ID NOs: 1-35in US Patent Publication No. 2009/0226470, SEQ ID NOs: 1-35 in US PatentPublication No. 2013/0177581, SEQ ID NOs: 1-35 in International PatentPublication No. WO2009/075886, SEQ ID NOs: 1-5, and 7-645 inInternational Patent Publication No. WO2012/009644, SEQ ID NO: 1 inInternational Patent Publication No. WO1999/024595, SEQ ID NO: 1 in U.S.Pat. No. 6,310,197, and SEQ ID NO: 1 in U.S. Pat. No. 6,849,405, the TEEsequences of each of which are incorporated herein by reference.

In yet another non-limiting example, the TEE may be an internal ribosomeentry site (IRES), HCV-IRES or an IRES element such as, but not limitedto, those described in U.S. Pat. No. 7,468,275, US Patent PublicationNos. 2007/0048776 and 2011/0124100 and International Patent PublicationNos. WO2007/025008 and WO2001/055369, the IRES sequences of each ofwhich are incorporated herein by reference. The IRES elements mayinclude, but are not limited to, the Gtx sequences (e.g., Gtx9-nt,Gtx8-nt, Gtx7-nt) described by Chappell et al. (Proc. Natl. Acad. Sci.USA 101:9590-9594, 2004) and Zhou et al. (PNAS 102:6273-6278, 2005) andin US Patent Publication Nos. 2007/0048776 and 2011/0124100 andInternational Patent Publication No. WO2007/025008, the IRES sequencesof each of which are incorporated herein by reference.

“Translational enhancer polynucleotides” are polynucleotides whichinclude one or more of the specific TEE exemplified herein and/ordisclosed in the art (see e.g., U.S. Pat. Nos. 6,310,197, 6,849,405,7,456,273, 7,183,395, U.S. Patent Publication Nos. 20090/226470,2007/0048776, 2011/0124100, 2009/0093049, 2013/0177581, InternationalPatent Publication Nos. WO2009/075886, WO2007/025008, WO2012/009644,WO2001/055371 WO1999/024595, and European Patent Nos. 2610341 and2610340; the TEE sequences of each of which are incorporated herein byreference) or their variants, homologs or functional derivatives. One ormultiple copies of a specific TEE can be present in a polynucleotide(e.g., mRNA). The TEEs in the translational enhancer polynucleotides canbe organized in one or more sequence segments. A sequence segment canharbor one or more of the specific TEEs exemplified herein, with eachTEE being present in one or more copies. When multiple sequence segmentsare present in a translational enhancer polynucleotide, they can behomogenous or heterogeneous. Thus, the multiple sequence segments in atranslational enhancer polynucleotide can harbor identical or differenttypes of the specific TEEs exemplified herein, identical or differentnumber of copies of each of the specific TEEs, and/or identical ordifferent organization of the TEEs within each sequence segment.

A polynucleotide (e.g., mRNA) may include at least one TEE that isdescribed in International Patent Publication Nos. WO1999/024595,WO2012/009644, WO2009/075886, WO2007/025008, WO1999/024595, EuropeanPatent Publication Nos. 2610341 and 2610340, U.S. Pat. Nos. 6,310,197,6,849,405, 7,456,273, 7,183,395, and US Patent Publication Nos.2009/0226470, 2011/0124100, 2007/0048776, 2009/0093049, and 2013/0177581the TEE sequences of each of which are incorporated herein by reference.The TEE may be located in the 5′-UTR of the polynucleotides (e.g.,mRNA).

A polynucleotide (e.g., mRNA) may include at least one TEE that has atleast 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95% or atleast 99% identity with the TEEs described in US Patent Publication Nos.2009/0226470, 2007/0048776, 2013/0177581 and 2011/0124100, InternationalPatent Publication Nos. WO1999/024595, WO2012/009644, WO2009/075886 andWO2007/025008, European Patent Publication Nos. 2610341 and 2610340,U.S. Pat. Nos. 6,310,197, 6,849,405, 7,456,273, 7,183,395, the TEEsequences of each of which are incorporated herein by reference.

The 5′-UTR of a polynucleotide (e.g., mRNA) may include at least 1, atleast 2, at least 3, at least 4, at least 5, at least 6, at least 7, atleast 8, at least 9, at least 10, at least 11, at least 12, at least 13,at least 14, at least 15, at least 16, at least 17, at least 18 at least19, at least 20, at least 21, at least 22, at least 23, at least 24, atleast 25, at least 30, at least 35, at least 40, at least 45, at least50, at least 55 or more than 60 TEE sequences. The TEE sequences in the5′-UTR of a polynucleotide (e.g., mRNA) may be the same or different TEEsequences. The TEE sequences may be in a pattern such as ABABAB,AABBAABBAABB, or ABCABCABC, or variants thereof, repeated once, twice,or more than three times. In these patterns, each letter, A, B, or Crepresent a different TEE sequence at the nucleotide level.

In some cases, the 5′-UTR may include a spacer to separate two TEEsequences. As a non-limiting example, the spacer may be a 15 nucleotidespacer and/or other spacers known in the art. As another non-limitingexample, the 5′-UTR may include a TEE sequence-spacer module repeated atleast once, at least twice, at least 3 times, at least 4 times, at least5 times, at least 6 times, at least 7 times, at least 8 times, at least9 times, or more than 9 times in the 5′-UTR.

In other instances, the spacer separating two TEE sequences may includeother sequences known in the art which may regulate the translation ofthe polynucleotides (e.g., mRNA) of the present disclosure such as, butnot limited to, miR sequences (e.g., miR binding sites and miR seeds).As a non-limiting example, each spacer used to separate two TEEsequences may include a different miR sequence or component of a miRsequence (e.g., miR seed sequence).

In some instances, the TEE in the 5′-UTR of a polynucleotide (e.g.,mRNA) may include at least 5%, at least 10%, at least 15%, at least 20%,at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, atleast 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 99% or more than 99% of the TEE sequences disclosed in US PatentPublication Nos. 2009/0226470, 2007/0048776, 2013/0177581 and2011/0124100, International Patent Publication Nos. WO 1999/024595,WO2012/009644, WO2009/075886 and WO2007/025008, European PatentPublication Nos. 2610341 and 2610340, and U.S. Pat. Nos. 6,310,197,6,849,405, 7,456,273, and 7,183,395 the TEE sequences of each of whichare incorporated herein by reference. In another embodiment, the TEE inthe 5′-UTR of the polynucleotides (e.g., mRNA) of the present disclosuremay include a 5-30 nucleotide fragment, a 5-25 nucleotide fragment, a5-20 nucleotide fragment, a 5-15 nucleotide fragment, a 5-10 nucleotidefragment of the TEE sequences disclosed in US Patent Publication Nos.2009/0226470, 2007/0048776, 2013/0177581 and 2011/0124100, InternationalPatent Publication Nos. WO1999/024595, WO2012/009644, WO2009/075886 andWO2007/025008, European Patent Publication Nos. 2610341 and 2610340, andU.S. Pat. Nos. 6,310,197, 6,849,405, 7,456,273, and 7,183,395; the TEEsequences of each of which are incorporated herein by reference.

In certain cases, the TEE in the 5′-UTR of the polynucleotides (e.g.,mRNA) of the present disclosure may include at least 5%, at least 10%,at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, atleast 40%, at least 45%, at least 50%, at least 55%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 99% or more than 99% of the TEEsequences disclosed in Chappell et al. (Proc. Natl. Acad. Sci. USA101:9590-9594, 2004) and Zhou et al. (PNAS 102:6273-6278, 2005), inSupplemental Table 1 and in Supplemental Table 2 disclosed by Wellensieket al (Genome-wide profiling of human cap-independenttranslation-enhancing elements, Nature Methods, 2013; DOI:10.1038/NMETH.2522); the TEE sequences of each of which are hereinincorporated by reference. In another embodiment, the TEE in the 5′-UTRof the polynucleotides (e.g., mRNA) of the present disclosure mayinclude a 5-30 nucleotide fragment, a 5-25 nucleotide fragment, a 5-20nucleotide fragment, a 5-15 nucleotide fragment, a 5-10 nucleotidefragment of the TEE sequences disclosed in Chappell et al. (Proc. Natl.Acad. Sci. USA 101:9590-9594, 2004) and Zhou et al. (PNAS 102:6273-6278,2005), in Supplemental Table 1 and in Supplemental Table 2 disclosed byWellensiek et al (Genome-wide profiling of human cap-independenttranslation-enhancing elements, Nature Methods, 2013;DOI:10.1038/NMETH.2522); the TEE sequences of each of which isincorporated herein by reference.

In some cases, the TEE used in the 5′-UTR of a polynucleotide (e.g.,mRNA) is an IRES sequence such as, but not limited to, those describedin U.S. Pat. No. 7,468,275 and International Patent Publication No.WO2001/055369, the TEE sequences of each of which are incorporatedherein by reference.

In some instances, the TEEs used in the 5′-UTR of a polynucleotide(e.g., mRNA) may be identified by the methods described in US PatentPublication Nos. 2007/0048776 and 2011/0124100 and International PatentPublication Nos. WO2007/025008 and WO2012/009644, the methods of each ofwhich are incorporated herein by reference.

In some cases, the TEEs used in the 5′-UTR of a polynucleotide (e.g.,mRNA) of the present disclosure may be a transcription regulatoryelement described in U.S. Pat. Nos. 7,456,273 and 7,183,395, US PatentPublication No. 2009/0093049, and International Publication No.WO2001/055371, the TEE sequences of each of which is incorporated hereinby reference. The transcription regulatory elements may be identified bymethods known in the art, such as, but not limited to, the methodsdescribed in U.S. Pat. Nos. 7,456,273 and 7,183,395, US PatentPublication No. 2009/0093049, and International Publication No.WO2001/055371, the methods of each of which is incorporated herein byreference.

In yet other instances, the TEE used in the 5′-UTR of a polynucleotide(e.g., mRNA) is a polynucleotide or portion thereof as described in U.S.Pat. Nos. 7,456,273 and 7,183,395, US Patent Publication No.2009/0093049, and International Publication No. WO2001/055371, the TEEsequences of each of which are incorporated herein by reference.

The 5′-UTR including at least one TEE described herein may beincorporated in a monocistronic sequence such as, but not limited to, avector system or a polynucleotide vector. As a non-limiting example, thevector systems and polynucleotide vectors may include those described inU.S. Pat. Nos. 7,456,273 and 7,183,395, US Patent Publication Nos.2007/0048776, 2009/0093049 and 2011/0124100, and International PatentPublication Nos. WO2007/025008 and WO2001/055371, the TEE sequences ofeach of which are incorporated herein by reference.

The TEEs described herein may be located in the 5′-UTR and/or the 3′-UTRof the polynucleotides (e.g., mRNA). The TEEs located in the 3′-UTR maybe the same and/or different than the TEEs located in and/or describedfor incorporation in the 5′-UTR.

In some cases, the 3′-UTR of a polynucleotide (e.g., mRNA) may includeat least 1, at least 2, at least 3, at least 4, at least 5, at least 6,at least 7, at least 8, at least 9, at least 10, at least 11, at least12, at least 13, at least 14, at least 15, at least 16, at least 17, atleast 18 at least 19, at least 20, at least 21, at least 22, at least23, at least 24, at least 25, at least 30, at least 35, at least 40, atleast 45, at least 50, at least 55 or more than 60 TEE sequences. TheTEE sequences in the 3′-UTR of the polynucleotides (e.g., mRNA) of thepresent disclosure may be the same or different TEE sequences. The TEEsequences may be in a pattern such as ABABAB, AABBAABBAABB, orABCABCABC, or variants thereof, repeated once, twice, or more than threetimes. In these patterns, each letter, A, B, or C represent a differentTEE sequence at the nucleotide level.

In one instance, the 3′-UTR may include a spacer to separate two TEEsequences. As a non-limiting example, the spacer may be a 15 nucleotidespacer and/or other spacers known in the art. As another non-limitingexample, the 3′-UTR may include a TEE sequence-spacer module repeated atleast once, at least twice, at least 3 times, at least 4 times, at least5 times, at least 6 times, at least 7 times, at least 8 times, at least9 times, or more than 9 times in the 3′-UTR.

In other cases, the spacer separating two TEE sequences may includeother sequences known in the art which may regulate the translation ofthe polynucleotides (e.g., mRNA) of the present disclosure such as, butnot limited to, miR sequences described herein (e.g., miR binding sitesand miR seeds). As a non-limiting example, each spacer used to separatetwo TEE sequences may include a different miR sequence or component of amiR sequence (e.g., miR seed sequence).

In yet other cases, the incorporation of a miR sequence and/or a TEEsequence changes the shape of the stem loop region which may increaseand/or decrease translation. (see e.g., Kedde et al. A Pumilio-inducedRNA structure switch in p27-3′UTR controls miR-221 and miR-22accessibility. Nature Cell Biology. 2010).

Stem Loops

Polynucleotides (e.g., mRNAs) may include a stem loop such as, but notlimited to, a histone stem loop. The stem loop may be a nucleotidesequence that is about 25 or about 26 nucleotides in length such as, butnot limited to, SEQ ID NOs: 7-17 as described in International PatentPublication No. WO2013/103659, of which SEQ ID NOs: 7-17 areincorporated herein by reference. The histone stem loop may be located3′-relative to the coding region (e.g., at the 3′-terminus of the codingregion). As a non-limiting example, the stem loop may be located at the3′-end of a polynucleotide described herein. In some cases, apolynucleotide (e.g., an mRNA) includes more than one stem loop (e.g.,two stem loops). Examples of stem loop sequences are described inInternational Patent Publication Nos. WO2012/019780 and WO201502667, thestem loop sequences of which are herein incorporated by reference. Insome instances, a polynucleotide includes the stem loop sequenceCAAAGGCTCTTTTCAGAGCCACCA (SEQ ID NO: 5). In others, a polynucleotideincludes the stem loop sequence CAAAGGCUCUUUUCAGAGCCACCA (SEQ ID NO: 6).

A stem loop may be located in a second terminal region of apolynucleotide. As a non-limiting example, the stem loop may be locatedwithin an untranslated region (e.g., 3′-UTR) in a second terminalregion.

In some cases, a polynucleotide such as, but not limited to mRNA, whichincludes the histone stem loop may be stabilized by the addition of a3′-stabilizing region (e.g., a 3′-stabilizing region including at leastone chain terminating nucleoside). Not wishing to be bound by theory,the addition of at least one chain terminating nucleoside may slow thedegradation of a polynucleotide and thus can increase the half-life ofthe polynucleotide.

In other cases, a polynucleotide such as, but not limited to mRNA, whichincludes the histone stem loop may be stabilized by an alteration to the3′-region of the polynucleotide that can prevent and/or inhibit theaddition of oligio(U) (see e.g., International Patent Publication No.WO2013/103659).

In yet other cases, a polynucleotide such as, but not limited to mRNA,which includes the histone stem loop may be stabilized by the additionof an oligonucleotide that terminates in a 3′-deoxynucleoside,2′,3′-dideoxynucleoside 3′-O-methylnucleosides, 3′-O-ethylnucleosides,3′-arabinosides, and other alternative nucleosides known in the artand/or described herein.

In some instances, the polynucleotides of the present disclosure mayinclude a histone stem loop, a poly-A region, and/or a 5′-cap structure.The histone stem loop may be before and/or after the poly-A region. Thepolynucleotides including the histone stem loop and a poly-A regionsequence may include a chain terminating nucleoside described herein.

In other instances, the polynucleotides of the present disclosure mayinclude a histone stem loop and a 5′-cap structure. The 5′-cap structuremay include, but is not limited to, those described herein and/or knownin the art.

In some cases, the conserved stem loop region may include a miR sequencedescribed herein. As a non-limiting example, the stem loop region mayinclude the seed sequence of a miR sequence described herein. In anothernon-limiting example, the stem loop region may include a miR-122 seedsequence.

In certain instances, the conserved stem loop region may include a miRsequence described herein and may also include a TEE sequence.

In some cases, the incorporation of a miR sequence and/or a TEE sequencechanges the shape of the stem loop region which may increase and/ordecrease translation. (See, e.g., Kedde et al. A Pumilio-induced RNAstructure switch in p27-3′UTR controls miR-221 and miR-22 accessibility.Nature Cell Biology. 2010, herein incorporated by reference in itsentirety).

Polynucleotides may include at least one histone stem-loop and a poly-Aregion or polyadenylation signal. Non-limiting examples ofpolynucleotide sequences encoding for at least one histone stem-loop anda poly-A region or a polyadenylation signal are described inInternational Patent Publication No. WO2013/120497, WO2013/120629,WO2013/120500, WO2013/120627, WO2013/120498, WO2013/120626,WO2013/120499 and WO2013/120628, the sequences of each of which areincorporated herein by reference. In certain cases, the polynucleotideencoding for a histone stem loop and a poly-A region or apolyadenylation signal may code for a pathogen antigen or fragmentthereof such as the polynucleotide sequences described in InternationalPatent Publication No WO2013/120499 and WO2013/120628, the sequences ofboth of which are incorporated herein by reference. In other cases, thepolynucleotide encoding for a histone stem loop and a poly-A region or apolyadenylation signal may code for a therapeutic protein such as thepolynucleotide sequences described in International Patent PublicationNo WO2013/120497 and WO2013/120629, the sequences of both of which areincorporated herein by reference. In some cases, the polynucleotideencoding for a histone stem loop and a poly-A region or apolyadenylation signal may code for a tumor antigen or fragment thereofsuch as the polynucleotide sequences described in International PatentPublication No WO2013/120500 and WO2013/120627, the sequences of both ofwhich are incorporated herein by reference. In other cases, thepolynucleotide encoding for a histone stem loop and a poly-A region or apolyadenylation signal may code for a allergenic antigen or anautoimmune self-antigen such as the polynucleotide sequences describedin International Patent Publication No WO2013/120498 and WO2013/120626,the sequences of both of which are incorporated herein by reference.

Poly-A Regions

A polynucleotide or nucleic acid (e.g., an mRNA) may include a polyAsequence and/or polyadenylation signal. A polyA sequence may becomprised entirely or mostly of adenine nucleotides or analogs orderivatives thereof. A polyA sequence may be a tail located adjacent toa 3′ untranslated region of a nucleic acid.

During RNA processing, a long chain of adenosine nucleotides (poly-Aregion) is normally added to messenger RNA (mRNA) molecules to increasethe stability of the molecule. Immediately after transcription, the3′-end of the transcript is cleaved to free a 3′-hydroxy. Then poly-Apolymerase adds a chain of adenosine nucleotides to the RNA. Theprocess, called polyadenylation, adds a poly-A region that is between100 and 250 residues long.

Unique poly-A region lengths may provide certain advantages to thealternative polynucleotides of the present disclosure.

Generally, the length of a poly-A region of the present disclosure is atleast 30 nucleotides in length. In another embodiment, the poly-A regionis at least 35 nucleotides in length. In another embodiment, the lengthis at least 40 nucleotides. In another embodiment, the length is atleast 45 nucleotides. In another embodiment, the length is at least 55nucleotides. In another embodiment, the length is at least 60nucleotides. In another embodiment, the length is at least 70nucleotides. In another embodiment, the length is at least 80nucleotides. In another embodiment, the length is at least 90nucleotides. In another embodiment, the length is at least 100nucleotides. In another embodiment, the length is at least 120nucleotides. In another embodiment, the length is at least 140nucleotides. In another embodiment, the length is at least 160nucleotides. In another embodiment, the length is at least 180nucleotides. In another embodiment, the length is at least 200nucleotides. In another embodiment, the length is at least 250nucleotides. In another embodiment, the length is at least 300nucleotides. In another embodiment, the length is at least 350nucleotides. In another embodiment, the length is at least 400nucleotides. In another embodiment, the length is at least 450nucleotides. In another embodiment, the length is at least 500nucleotides. In another embodiment, the length is at least 600nucleotides. In another embodiment, the length is at least 700nucleotides. In another embodiment, the length is at least 800nucleotides. In another embodiment, the length is at least 900nucleotides. In another embodiment, the length is at least 1000nucleotides. In another embodiment, the length is at least 1100nucleotides. In another embodiment, the length is at least 1200nucleotides. In another embodiment, the length is at least 1300nucleotides. In another embodiment, the length is at least 1400nucleotides. In another embodiment, the length is at least 1500nucleotides. In another embodiment, the length is at least 1600nucleotides. In another embodiment, the length is at least 1700nucleotides. In another embodiment, the length is at least 1800nucleotides. In another embodiment, the length is at least 1900nucleotides. In another embodiment, the length is at least 2000nucleotides. In another embodiment, the length is at least 2500nucleotides. In another embodiment, the length is at least 3000nucleotides.

In some instances, the poly-A region may be 80 nucleotides, 120nucleotides, 160 nucleotides in length on an alternative polynucleotidemolecule described herein.

In other instances, the poly-A region may be 20, 40, 80, 100, 120, 140or 160 nucleotides in length on an alternative polynucleotide moleculedescribed herein.

In some cases, the poly-A region is designed relative to the length ofthe overall alternative polynucleotide. This design may be based on thelength of the coding region of the alternative polynucleotide, thelength of a particular feature or region of the alternativepolynucleotide (such as mRNA), or based on the length of the ultimateproduct expressed from the alternative polynucleotide. When relative toany feature of the alternative polynucleotide (e.g., other than the mRNAportion which includes the poly-A region) the poly-A region may be 10,20, 30, 40, 50, 60, 70, 80, 90 or 100% greater in length than theadditional feature. The poly-A region may also be designed as a fractionof the alternative polynucleotide to which it belongs. In this context,the poly-A region may be 10, 20, 30, 40, 50, 60, 70, 80, or 90% or moreof the total length of the construct or the total length of theconstruct minus the poly-A region.

In certain cases, engineered binding sites and/or the conjugation ofpolynucleotides (e.g., mRNA) for poly-A binding protein may be used toenhance expression. The engineered binding sites may be sensor sequenceswhich can operate as binding sites for ligands of the localmicroenvironment of the polynucleotides (e.g., mRNA). As a non-limitingexample, the polynucleotides (e.g., mRNA) may include at least oneengineered binding site to alter the binding affinity of poly-A bindingprotein (PABP) and analogs thereof. The incorporation of at least oneengineered binding site may increase the binding affinity of the PABPand analogs thereof.

Additionally, multiple distinct polynucleotides (e.g., mRNA) may belinked together to the PABP (poly-A binding protein) through the 3′-endusing alternative nucleotides at the 3′-terminus of the poly-A region.Transfection experiments can be conducted in relevant cell lines at andprotein production can be assayed by ELISA at 12 hours, 24 hours, 48hours, 72 hours, and day 7 post-transfection. As a non-limiting example,the transfection experiments may be used to evaluate the effect on PABPor analogs thereof binding affinity as a result of the addition of atleast one engineered binding site.

In certain cases, a poly-A region may be used to modulate translationinitiation. While not wishing to be bound by theory, the poly-A regionrecruits PABP which in turn can interact with translation initiationcomplex and thus may be essential for protein synthesis.

In some cases, a poly-A region may also be used in the presentdisclosure to protect against 3′-5′-exonuclease digestion.

In some instances, a polynucleotide (e.g., mRNA) may include a polyA-GQuartet. The G-quartet is a cyclic hydrogen bonded array of fourguanosine nucleotides that can be formed by G-rich sequences in both DNAand RNA. In this embodiment, the G-quartet is incorporated at the end ofthe poly-A region. The resultant polynucleotides (e.g., mRNA) may beassayed for stability, protein production and other parameters includinghalf-life at various time points. It has been discovered that thepolyA-G quartet results in protein production equivalent to at least 75%of that seen using a poly-A region of 120 nucleotides alone.

In some cases, a polynucleotide (e.g., mRNA) may include a poly-A regionand may be stabilized by the addition of a 3′-stabilizing region. Thepolynucleotides (e.g., mRNA) with a poly-A region may further include a5′-cap structure.

In other cases, a polynucleotide (e.g., mRNA) may include a poly-A-GQuartet. The polynucleotides (e.g., mRNA) with a poly-A-G Quartet mayfurther include a 5′-cap structure.

In some cases, the 3′-stabilizing region which may be used to stabilizea polynucleotide (e.g., mRNA) including a poly-A region or poly-A-GQuartet may be, but is not limited to, those described in InternationalPatent Publication No. WO2013/103659, the poly-A regions and poly-A-GQuartets of which are incorporated herein by reference. In other cases,the 3′-stabilizing region which may be used with the present disclosureinclude a chain termination nucleoside such as 3′-deoxyadenosine(cordycepin), 3′-deoxyuridine, 3′-deoxycytosine, 3′-deoxyguanosine,3′-deoxythymine, 2′,3′-dideoxynucleosides, such as2′,3′-dideoxyadenosine, 2′,3′-dideoxyuridine, 2′,3′-dideoxycytosine,2′,3′-dideoxyguanosine, 2′,3′-dideoxythymine, a 2′-deoxynucleoside, oran O-methylnucleoside.

In other cases, a polynucleotide such as, but not limited to mRNA, whichincludes a polyA region or a poly-A-G Quartet may be stabilized by analteration to the 3′-region of the polynucleotide that can preventand/or inhibit the addition of oligio(U) (see e.g., International PatentPublication No. WO2013/103659).

In yet other instances, a polynucleotide such as, but not limited tomRNA, which includes a poly-A region or a poly-A-G Quartet may bestabilized by the addition of an oligonucleotide that terminates in a3′-deoxynucleoside, 2′,3′-dideoxynucleoside 3′-O-methylnucleosides,3′-O-ethylnucleosides, 3′-arabinosides, and other alternativenucleosides known in the art and/or described herein.

Chain Terminating Nucleosides

A nucleic acid may include a chain terminating nucleoside. For example,a chain terminating nucleoside may include those nucleosidesdeoxygenated at the 2′ and/or 3′ positions of their sugar group. Suchspecies may include 3′-deoxyadenosine (cordycepin), 3′-deoxyuridine,3′-deoxycytosine, 3′-deoxyguanosine, 3′-deoxythymine, and2′,3′-dideoxynucleosides, such as 2′,3′-dideoxyadenosine,2′,3′-dideoxyuridine, 2′,3′-dideoxycytosine, 2′,3′-dideoxyguanosine, and2′,3′-dideoxythymine.

Other Components

A nanoparticle composition may include one or more components inaddition to those described in the preceding sections. For example, ananoparticle composition may include one or more small hydrophobicmolecules such as a vitamin (e.g., vitamin A or vitamin E) or a sterol.

Nanoparticle compositions may also include one or more permeabilityenhancer molecules, carbohydrates, polymers, surface altering agents, orother components. A permeability enhancer molecule may be a moleculedescribed by U.S. patent application publication No. 2005/0222064, forexample. Carbohydrates may include simple sugars (e.g., glucose) andpolysaccharides (e.g., glycogen and derivatives and analogs thereof).

A polymer may be included in and/or used to encapsulate or partiallyencapsulate a nanoparticle composition. A polymer may be biodegradableand/or biocompatible. A polymer may be selected from, but is not limitedto, polyamines, polyethers, polyamides, polyesters, polycarbamates,polyureas, polycarbonates, polystyrenes, polyimides, polysulfones,polyurethanes, polyacetylenes, polyethylenes, polyethyleneimines,polyisocyanates, polyacrylates, polymethacrylates, polyacrylonitriles,and polyarylates. For example, a polymer may include poly(caprolactone)(PCL), ethylene vinyl acetate polymer (EVA), poly(lactic acid) (PLA),poly(L-lactic acid) (PLLA), poly(glycolic acid) (PGA), poly(lacticacid-co-glycolic acid) (PLGA), poly(L-lactic acid-co-glycolic acid)(PLLGA), poly(D,L-lactide) (PDLA), poly(L-lactide) (PLLA),poly(D,L-lactide-co-caprolactone),poly(D,L-lactide-co-caprolactone-co-glycolide),poly(D,L-lactide-co-PEO-co-D,L-lactide),poly(D,L-lactide-co-PPO-co-D,L-lactide), polyalkyl cyanoacrylate,polyurethane, poly-L-lysine (PLL), hydroxypropyl methacrylate (HPMA),polyethyleneglycol, poly-L-glutamic acid, poly(hydroxy acids),polyanhydrides, polyorthoesters, poly(ester amides), polyamides,poly(ester ethers), polycarbonates, polyalkylenes such as polyethyleneand polypropylene, polyalkylene glycols such as poly(ethylene glycol)(PEG), polyalkylene oxides (PEO), polyalkylene terephthalates such aspoly(ethylene terephthalate), polyvinyl alcohols (PVA), polyvinylethers, polyvinyl esters such as poly(vinyl acetate), polyvinyl halidessuch as poly(vinyl chloride) (PVC), polyvinylpyrrolidone (PVP),polysiloxanes, polystyrene (PS), polyurethanes, derivatized cellulosessuch as alkyl celluloses, hydroxyalkyl celluloses, cellulose ethers,cellulose esters, nitro celluloses, hydroxypropylcellulose,carboxymethylcellulose, polymers of acrylic acids, such aspoly(methyl(meth)acrylate) (PMMA), poly(ethyl(meth)acrylate),poly(butyl(meth)acrylate), poly(isobutyl(meth)acrylate),poly(hexyl(meth)acrylate), poly(isodecyl(meth)acrylate),poly(lauryl(meth)acrylate), poly(phenyl(meth)acrylate), poly(methylacrylate), poly(isopropyl acrylate), poly(isobutyl acrylate),poly(octadecyl acrylate) and copolymers and mixtures thereof,polydioxanone and its copolymers, polyhydroxyalkanoates, polypropylenefumarate, polyoxymethylene, poloxamers, polyoxamines, poly(ortho)esters,poly(butyric acid), poly(valeric acid), poly(lactide-co-caprolactone),trimethylene carbonate, poly(N-acryloylmorpholine) (PAcM),poly(2-methyl-2-oxazoline) (PMOX), poly(2-ethyl-2-oxazoline) (PEOZ), andpolyglycerol.

Surface altering agents may include, but are not limited to, anionicproteins (e.g., bovine serum albumin), surfactants (e.g., cationicsurfactants such as dimethyldioctadecyl-ammonium bromide), sugars orsugar derivatives (e.g., cyclodextrin), nucleic acids, polymers (e.g.,heparin, polyethylene glycol, and poloxamer), mucolytic agents (e.g.,acetylcysteine, mugwort, bromelain, papain, clerodendrum, bromhexine,carbocisteine, eprazinone, mesna, ambroxol, sobrerol, domiodol,letosteine, stepronin, tiopronin, gelsolin, thymosin β4, dornase alfa,neltenexine, and erdosteine), and DNases (e.g., rhDNase). A surfacealtering agent may be disposed within a nanoparticle and/or on thesurface of a nanoparticle composition (e.g., by coating, adsorption,covalent linkage, or other process).

A nanoparticle composition may also comprise one or more functionalizedlipids. For example, a lipid may be functionalized with an alkyne groupthat, when exposed to an azide under appropriate reaction conditions,may undergo a cycloaddition reaction. In particular, a lipid bilayer maybe functionalized in this fashion with one or more groups useful infacilitating membrane permeation, cellular recognition, or imaging. Thesurface of a nanoparticle composition may also be conjugated with one ormore useful antibodies. Functional groups and conjugates useful intargeted cell delivery, imaging, and membrane permeation are well knownin the art.

In addition to these components, nanoparticle compositions may includeany substance useful in pharmaceutical compositions. For example, thenanoparticle composition may include one or more pharmaceuticallyacceptable excipients or accessory ingredients such as, but not limitedto, one or more solvents, dispersion media, diluents, dispersion aids,suspension aids, granulating aids, disintegrants, fillers, glidants,liquid vehicles, binders, surface active agents, isotonic agents,thickening or emulsifying agents, buffering agents, lubricating agents,oils, preservatives, and other species. Excipients such as waxes,butters, coloring agents, coating agents, flavorings, and perfumingagents may also be included. Pharmaceutically acceptable excipients arewell known in the art (see for example Remington's The Science andPractice of Pharmacy, 21^(st) Edition, A. R. Gennaro; Lippincott,Williams & Wilkins, Baltimore, Md., 2006).

Examples of diluents may include, but are not limited to, calciumcarbonate, sodium carbonate, calcium phosphate, dicalcium phosphate,calcium sulfate, calcium hydrogen phosphate, sodium phosphate lactose,sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol,sorbitol, inositol, sodium chloride, dry starch, cornstarch, powderedsugar, and/or combinations thereof. Granulating and dispersing agentsmay be selected from the non-limiting list consisting of potato starch,corn starch, tapioca starch, sodium starch glycolate, clays, alginicacid, guar gum, citrus pulp, agar, bentonite, cellulose and woodproducts, natural sponge, cation-exchange resins, calcium carbonate,silicates, sodium carbonate, cross-linked poly(vinyl-pyrrolidone)(crospovidone), sodium carboxymethyl starch (sodium starch glycolate),carboxymethyl cellulose, cross-linked sodium carboxymethyl cellulose(croscarmellose), methylcellulose, pregelatinized starch (starch 1500),microcrystalline starch, water insoluble starch, calcium carboxymethylcellulose, magnesium aluminum silicate (VEEGUM®), sodium lauryl sulfate,quaternary ammonium compounds, and/or combinations thereof.

Surface active agents and/or emulsifiers may include, but are notlimited to, natural emulsifiers (e.g. acacia, agar, alginic acid, sodiumalginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin,egg yolk, casein, wool fat, cholesterol, wax, and lecithin), colloidalclays (e.g. bentonite [aluminum silicate] and VEEGUM® [magnesiumaluminum silicate]), long chain amino acid derivatives, high molecularweight alcohols (e.g. stearyl alcohol, cetyl alcohol, oleyl alcohol,triacetin monostearate, ethylene glycol distearate, glycerylmonostearate, and propylene glycol monostearate, polyvinyl alcohol),carbomers (e.g. carboxy polymethylene, polyacrylic acid, acrylic acidpolymer, and carboxyvinyl polymer), carrageenan, cellulosic derivatives(e.g. carboxymethylcellulose sodium, powdered cellulose, hydroxymethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose,methylcellulose), sorbitan fatty acid esters (e.g. polyoxyethylenesorbitan monolaurate [TWEEN®20], polyoxyethylene sorbitan [TWEEN® 60],polyoxyethylene sorbitan monooleate [TWEEN®80], sorbitan monopalmitate[SPAN®40], sorbitan monostearate [SPAN®60], sorbitan tristearate[SPAN®65], glyceryl monooleate, sorbitan monooleate [SPAN®80]),polyoxyethylene esters (e.g. polyoxyethylene monostearate [MYRJ® 45],polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil,polyoxymethylene stearate, and SOLUTOL®), sucrose fatty acid esters,polyethylene glycol fatty acid esters (e.g. CREMOPHOR®), polyoxyethyleneethers, (e.g. polyoxyethylene lauryl ether [BRIJ® 30]),poly(vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamineoleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyllaurate, sodium lauryl sulfate, PLURONIC®F 68, POLOXAMER® 188,cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride,docusate sodium, and/or combinations thereof.

A binding agent may be starch (e.g. cornstarch and starch paste);gelatin; sugars (e.g. sucrose, glucose, dextrose, dextrin, molasses,lactose, lactitol, mannitol); natural and synthetic gums (e.g., acacia,sodium alginate, extract of Irish moss, panwar gum, ghatti gum, mucilageof isapol husks, carboxymethylcellulose, methylcellulose,ethylcellulose, hydroxyethylcellulose, hydroxypropyl cellulose,hydroxypropyl methylcellulose, microcrystalline cellulose, celluloseacetate, poly(vinyl-pyrrolidone), magnesium aluminum silicate (VEEGUM®),and larch arabogalactan); alginates; polyethylene oxide; polyethyleneglycol; inorganic calcium salts; silicic acid; polymethacrylates; waxes;water; alcohol; and combinations thereof, or any other suitable bindingagent.

Examples of preservatives may include, but are not limited to,antioxidants, chelating agents, antimicrobial preservatives, antifungalpreservatives, alcohol preservatives, acidic preservatives, and/or otherpreservatives. Examples of antioxidants include, but are not limited to,alpha tocopherol, ascorbic acid, acorbyl palmitate, butylatedhydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassiummetabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodiumbisulfite, sodium metabisulfite, and/or sodium sulfite. Examples ofchelating agents include ethylenediaminetetraacetic acid (EDTA), citricacid monohydrate, disodium edetate, dipotassium edetate, edetic acid,fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaricacid, and/or trisodium edetate. Examples of antimicrobial preservativesinclude, but are not limited to, benzalkonium chloride, benzethoniumchloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride,chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethylalcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol,phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and/orthimerosal. Examples of antifungal preservatives include, but are notlimited to, butyl paraben, methyl paraben, ethyl paraben, propylparaben, benzoic acid, hydroxybenzoic acid, potassium benzoate,potassium sorbate, sodium benzoate, sodium propionate, and/or sorbicacid. Examples of alcohol preservatives include, but are not limited to,ethanol, polyethylene glycol, benzyl alcohol, phenol, phenoliccompounds, bisphenol, chlorobutanol, hydroxybenzoate, and/or phenylethylalcohol. Examples of acidic preservatives include, but are not limitedto, vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, aceticacid, dehydroascorbic acid, ascorbic acid, sorbic acid, and/or phyticacid. Other preservatives include, but are not limited to, tocopherol,tocopherol acetate, deteroxime mesylate, cetrimide, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), ethylenediamine,sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), sodiumbisulfite, sodium metabisulfite, potassium sulfite, potassiummetabisulfite, GLYDANT PLUS®, PHENONIP®, methylparaben, GERMALL® 115,GERMABEN®II, NEOLONE™, KATHON™, and/or EUXYL®.

Examples of buffering agents include, but are not limited to, citratebuffer solutions, acetate buffer solutions, phosphate buffer solutions,ammonium chloride, calcium carbonate, calcium chloride, calcium citrate,calcium glubionate, calcium gluceptate, calcium gluconate, d-gluconicacid, calcium glycerophosphate, calcium lactate, calcium lactobionate,propanoic acid, calcium levulinate, pentanoic acid, dibasic calciumphosphate, phosphoric acid, tribasic calcium phosphate, calciumhydroxide phosphate, potassium acetate, potassium chloride, potassiumgluconate, potassium mixtures, dibasic potassium phosphate, monobasicpotassium phosphate, potassium phosphate mixtures, sodium acetate,sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate,dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphatemixtures, tromethamine, amino-sulfonate buffers (e.g., HEPES), magnesiumhydroxide, aluminum hydroxide, alginic acid, pyrogen-free water,isotonic saline, Ringer's solution, ethyl alcohol, and/or combinationsthereof. Lubricating agents may selected from the non-limiting groupconsisting of magnesium stearate, calcium stearate, stearic acid,silica, talc, malt, glyceryl behenate, hydrogenated vegetable oils,polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride,leucine, magnesium lauryl sulfate, sodium lauryl sulfate, andcombinations thereof.

Examples of oils include, but are not limited to, almond, apricotkernel, avocado, babassu, bergamot, black current seed, borage, cade,camomile, canola, caraway, carnauba, castor, cinnamon, cocoa butter,coconut, cod liver, coffee, corn, cotton seed, emu, eucalyptus, eveningprimrose, fish, flaxseed, geraniol, gourd, grape seed, hazel nut,hyssop, isopropyl myristate, jojoba, kukui nut, lavandin, lavender,lemon, litsea cubeba, macademia nut, mallow, mango seed, meadowfoamseed, mink, nutmeg, olive, orange, orange roughy, palm, palm kernel,peach kernel, peanut, poppy seed, pumpkin seed, rapeseed, rice bran,rosemary, safflower, sandalwood, sasquana, savoury, sea buckthorn,sesame, shea butter, silicone, soybean, sunflower, tea tree, thistle,tsubaki, vetiver, walnut, and wheat germ oils as well as butyl stearate,caprylic triglyceride, capric triglyceride, cyclomethicone, diethylsebacate, dimethicone 360, simethicone, isopropyl myristate, mineraloil, octyldodecanol, oleyl alcohol, silicone oil, and/or combinationsthereof.

Formulations

Nanoparticle compositions may include a lipid component and one or moreadditional components, such as a therapeutic and/or prophylactic. Ananoparticle composition may be designed for one or more specificapplications or targets. The elements of a nanoparticle composition maybe selected based on a particular application or target, and/or based onthe efficacy, toxicity, expense, ease of use, availability, or otherfeature of one or more elements. Similarly, the particular formulationof a nanoparticle composition may be selected for a particularapplication or target according to, for example, the efficacy andtoxicity of particular combinations of elements.

The lipid component of a nanoparticle composition may include, forexample, a lipid according to Formula (I), (IA), (II), (IIa), (IIb),(IIc), (IId) or (IIe), a phospholipid (such as an unsaturated lipid,e.g., DOPE or DSPC), a PEG lipid, and a structural lipid. The elementsof the lipid component may be provided in specific fractions.

In some embodiments, the lipid component of a nanoparticle compositionincludes a lipid according to Formula (I), (IA), (II), (IIa), (IIb),(IIc), (IId) or (IIe), a phospholipid, a PEG lipid, and a structurallipid. In certain embodiments, the lipid component of the nanoparticlecomposition includes about 30 mol % to about 60 mol % compound ofFormula (I), (IA), (II), (IIa), (IIb), (IIc), (IId) or (IIe), about 0mol % to about 30 mol % phospholipid, about 18.5 mol % to about 48.5 mol% structural lipid, and about 0 mol % to about 10 mol % of PEG lipid,provided that the total mol % does not exceed 100%. In some embodiments,the lipid component of the nanoparticle composition includes about 35mol % to about 55 mol % compound of Formula (I), (IA), (II), (IIa),(IIb), (IIc), (IId) or (IIe), about 5 mol % to about 25 mol %phospholipid, about 30 mol % to about 40 mol % structural lipid, andabout 0 mol % to about 10 mol % of PEG lipid. In a particularembodiment, the lipid component includes about 50 mol % said compound,about 10 mol % phospholipid, about 38.5 mol % structural lipid, andabout 1.5 mol % of PEG lipid. In another particular embodiment, thelipid component includes about 40 mol % said compound, about 20 mol %phospholipid, about 38.5 mol % structural lipid, and about 1.5 mol % ofPEG lipid. In some embodiments, the phospholipid may be DOPE or DSPC. Inother embodiments, the PEG lipid may be PEG-DMG and/or the structurallipid may be cholesterol.

Nanoparticle compositions may be designed for one or more specificapplications or targets. For example, a nanoparticle composition may bedesigned to deliver a therapeutic and/or prophylactic such as an RNA toa particular cell, tissue, organ, or system or group thereof in amammal's body. Physiochemical properties of nanoparticle compositionsmay be altered in order to increase selectivity for particular bodilytargets. For instance, particle sizes may be adjusted based on thefenestration sizes of different organs. The therapeutic and/orprophylactic included in a nanoparticle composition may also be selectedbased on the desired delivery target or targets. For example, atherapeutic and/or prophylactic may be selected for a particularindication, condition, disease, or disorder and/or for delivery to aparticular cell, tissue, organ, or system or group thereof (e.g.,localized or specific delivery). In certain embodiments, a nanoparticlecomposition may include an mRNA encoding a polypeptide of interestcapable of being translated within a cell to produce the polypeptide ofinterest. Such a composition may be designed to be specificallydelivered to a particular organ. In some embodiments, a composition maybe designed to be specifically delivered to a mammalian liver.

The amount of a therapeutic and/or prophylactic in a nanoparticlecomposition may depend on the size, composition, desired target and/orapplication, or other properties of the nanoparticle composition as wellas on the properties of the therapeutic and/or prophylactic. Forexample, the amount of an RNA useful in a nanoparticle composition maydepend on the size, sequence, and other characteristics of the RNA. Therelative amounts of a therapeutic and/or prophylactic and other elements(e.g., lipids) in a nanoparticle composition may also vary. In someembodiments, the wt/wt ratio of the lipid component to a therapeuticand/or prophylactic in a nanoparticle composition may be from about 5:1to about 60:1, such as 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1,14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1,50:1, and 60:1. For example, the wt/wt ratio of the lipid component to atherapeutic and/or prophylactic may be from about 10:1 to about 40:1. Incertain embodiments, the wt/wt ratio is about 20:1. The amount of atherapeutic and/or prophylactic in a nanoparticle composition may, forexample, be measured using absorption spectroscopy (e.g.,ultraviolet-visible spectroscopy).

In some embodiments, a nanoparticle composition includes one or moreRNAs, and the one or more RNAs, lipids, and amounts thereof may beselected to provide a specific N:P ratio. The N:P ratio of thecomposition refers to the molar ratio of nitrogen atoms in one or morelipids to the number of phosphate groups in an RNA. In general, a lowerN:P ratio is preferred. The one or more RNA, lipids, and amounts thereofmay be selected to provide an N:P ratio from about 2:1 to about 30:1,such as 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 12:1, 14:1, 16:1,18:1, 20:1, 22:1, 24:1, 26:1, 28:1, or 30:1. In certain embodiments, theN:P ratio may be from about 2:1 to about 8:1. In other embodiments, theN:P ratio is from about 5:1 to about 8:1. For example, the N:P ratio maybe about 5.0:1, about 5.5:1, about 5.67:1, about 6.0:1, about 6.5:1, orabout 7.0:1. For example, the N:P ratio may be about 5.67:1.

Physical Properties

The characteristics of a nanoparticle composition may depend on thecomponents thereof. For example, a nanoparticle composition includingcholesterol as a structural lipid may have different characteristicsthan a nanoparticle composition that includes a different structurallipid. Similarly, the characteristics of a nanoparticle composition maydepend on the absolute or relative amounts of its components. Forinstance, a nanoparticle composition including a higher molar fractionof a phospholipid may have different characteristics than a nanoparticlecomposition including a lower molar fraction of a phospholipid.Characteristics may also vary depending on the method and conditions ofpreparation of the nanoparticle composition.

Nanoparticle compositions may be characterized by a variety of methods.For example, microscopy (e.g., transmission electron microscopy orscanning electron microscopy) may be used to examine the morphology andsize distribution of a nanoparticle composition. Dynamic lightscattering or potentiometry (e.g., potentiometric titrations) may beused to measure zeta potentials. Dynamic light scattering may also beutilized to determine particle sizes. Instruments such as the ZetasizerNano ZS (Malvern Instruments Ltd, Malvern, Worcestershire, UK) may alsobe used to measure multiple characteristics of a nanoparticlecomposition, such as particle size, polydispersity index, and zetapotential.

The mean size of a nanoparticle composition may be between 10 s of nmand 100 s of nm, e.g., measured by dynamic light scattering (DLS). Forexample, the mean size may be from about 40 nm to about 150 nm, such asabout 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130nm, 135 nm, 140 nm, 145 nm, or 150 nm. In some embodiments, the meansize of a nanoparticle composition may be from about 50 nm to about 100nm, from about 50 nm to about 90 nm, from about 50 nm to about 80 nm,from about 50 nm to about 70 nm, from about 50 nm to about 60 nm, fromabout 60 nm to about 100 nm, from about 60 nm to about 90 nm, from about60 nm to about 80 nm, from about 60 nm to about 70 nm, from about 70 nmto about 100 nm, from about 70 nm to about 90 nm, from about 70 nm toabout 80 nm, from about 80 nm to about 100 nm, from about 80 nm to about90 nm, or from about 90 nm to about 100 nm. In certain embodiments, themean size of a nanoparticle composition may be from about 70 nm to about100 nm. In a particular embodiment, the mean size may be about 80 nm. Inother embodiments, the mean size may be about 100 nm.

A nanoparticle composition may be relatively homogenous. Apolydispersity index may be used to indicate the homogeneity of ananoparticle composition, e.g., the particle size distribution of thenanoparticle compositions. A small (e.g., less than 0.3) polydispersityindex generally indicates a narrow particle size distribution. Ananoparticle composition may have a polydispersity index from about 0 toabout 0.25, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08,0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20,0.21, 0.22, 0.23, 0.24, or 0.25. In some embodiments, the polydispersityindex of a nanoparticle composition may be from about 0.10 to about0.20.

The zeta potential of a nanoparticle composition may be used to indicatethe electrokinetic potential of the composition. For example, the zetapotential may describe the surface charge of a nanoparticle composition.Nanoparticle compositions with relatively low charges, positive ornegative, are generally desirable, as more highly charged species mayinteract undesirably with cells, tissues, and other elements in thebody. In some embodiments, the zeta potential of a nanoparticlecomposition may be from about −10 mV to about +20 mV, from about −10 mVto about +15 mV, from about −10 mV to about +10 mV, from about −10 mV toabout +5 mV, from about −10 mV to about 0 mV, from about −10 mV to about−5 mV, from about −5 mV to about +20 mV, from about −5 mV to about +15mV, from about −5 mV to about +10 mV, from about −5 mV to about +5 mV,from about −5 mV to about 0 mV, from about 0 mV to about +20 mV, fromabout 0 mV to about +15 mV, from about 0 mV to about +10 mV, from about0 mV to about +5 mV, from about +5 mV to about +20 mV, from about +5 mVto about +15 mV, or from about +5 mV to about +10 mV.

The efficiency of encapsulation of a therapeutic and/or prophylacticdescribes the amount of therapeutic and/or prophylactic that isencapsulated or otherwise associated with a nanoparticle compositionafter preparation, relative to the initial amount provided. Theencapsulation efficiency is desirably high (e.g., close to 100%). Theencapsulation efficiency may be measured, for example, by comparing theamount of therapeutic and/or prophylactic in a solution containing thenanoparticle composition before and after breaking up the nanoparticlecomposition with one or more organic solvents or detergents.Fluorescence may be used to measure the amount of free therapeuticand/or prophylactic (e.g., RNA) in a solution. For the nanoparticlecompositions described herein, the encapsulation efficiency of atherapeutic and/or prophylactic may be at least 50%, for example 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100%. In some embodiments, the encapsulationefficiency may be at least 80%. In certain embodiments, theencapsulation efficiency may be at least 90%.

A nanoparticle composition may optionally comprise one or more coatings.For example, a nanoparticle composition may be formulated in a capsule,film, or tablet having a coating. A capsule, film, or tablet including acomposition described herein may have any useful size, tensile strength,hardness, or density.

Pharmaceutical Compositions

Nanoparticle compositions may be formulated in whole or in part aspharmaceutical compositions. Pharmaceutical compositions may include oneor more nanoparticle compositions. For example, a pharmaceuticalcomposition may include one or more nanoparticle compositions includingone or more different therapeutic and/or prophylactics. Pharmaceuticalcompositions may further include one or more pharmaceutically acceptableexcipients or accessory ingredients such as those described herein.General guidelines for the formulation and manufacture of pharmaceuticalcompositions and agents are available, for example, in Remington's TheScience and Practice of Pharmacy, 21^(st) Edition, A. R. Gennaro;Lippincott, Williams & Wilkins, Baltimore, Md., 2006. Conventionalexcipients and accessory ingredients may be used in any pharmaceuticalcomposition, except insofar as any conventional excipient or accessoryingredient may be incompatible with one or more components of ananoparticle composition. An excipient or accessory ingredient may beincompatible with a component of a nanoparticle composition if itscombination with the component may result in any undesirable biologicaleffect or otherwise deleterious effect.

In some embodiments, one or more excipients or accessory ingredients maymake up greater than 50% of the total mass or volume of a pharmaceuticalcomposition including a nanoparticle composition. For example, the oneor more excipients or accessory ingredients may make up 50%, 60%, 70%,80%, 90%, or more of a pharmaceutical convention. In some embodiments, apharmaceutically acceptable excipient is at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% pure. In someembodiments, an excipient is approved for use in humans and forveterinary use. In some embodiments, an excipient is approved by UnitedStates Food and Drug Administration. In some embodiments, an excipientis pharmaceutical grade. In some embodiments, an excipient meets thestandards of the United States Pharmacopoeia (USP), the EuropeanPharmacopoeia (EP), the British Pharmacopoeia, and/or the InternationalPharmacopoeia.

Relative amounts of the one or more nanoparticle compositions, the oneor more pharmaceutically acceptable excipients, and/or any additionalingredients in a pharmaceutical composition in accordance with thepresent disclosure will vary, depending upon the identity, size, and/orcondition of the subject treated and further depending upon the route bywhich the composition is to be administered. By way of example, apharmaceutical composition may comprise between 0.1% and 100% (wt/wt) ofone or more nanoparticle compositions.

In certain embodiments, the nanoparticle compositions and/orpharmaceutical compositions of the disclosure are refrigerated or frozenfor storage and/or shipment (e.g., being stored at a temperature of 4°C. or lower, such as a temperature between about −150° C. and about 0°C. or between about −80° C. and about −20° C. (e.g., about −5° C., −10°C., −15° C., −20° C., −25° C., −30° C., −40° C., −50° C., −60° C., −70°C., −80° C., −90° C., −130° C. or −150° C.). For example, thepharmaceutical composition comprising a compound of any of Formulae (I),(IA), (II), and (IIa)-(IIe) is a solution that is refrigerated forstorage and/or shipment at, for example, about −20° C., −30° C., −40°C., −50° C., −60° C., −70° C., or −80° C. In certain embodiments, thedisclosure also relates to a method of increasing stability of thenanoparticle compositions and/or pharmaceutical compositions comprisinga compound of any of Formulae (I), (IA), (II), and (IIa)-(IIe) bystoring the nanoparticle compositions and/or pharmaceutical compositionsat a temperature of 4° C. or lower, such as a temperature between about−150° C. and about 0° C. or between about −80° C. and about −20° C.,e.g., about −5° C., −10° C., −15° C., −20° C., −25° C., −30° C., −40°C., −50° C., −60° C., −70° C., −80° C., −90° C., −130° C. or −150° C.).For example, the nanoparticle compositions and/or pharmaceuticalcompositions disclosed herein are stable for about at least 1 week, atleast 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, atleast 6 weeks, at least 1 month, at least 2 months, at least 4 months,at least 6 months, at least 8 months, at least 10 months, at least 12months, at least 14 months, at least 16 months, at least 18 months, atleast 20 months, at least 22 months, or at least 24 months, e.g., at atemperature of 4° C. or lower (e.g., between about 4° C. and −20° C.).In one embodiment, the formulation is stabilized for at least 4 weeks atabout 4° C. In certain embodiments, the pharmaceutical composition ofthe disclosure comprises a nanoparticle composition disclosed herein anda pharmaceutically acceptable carrier selected from one or more of Tris,an acetate (e.g., sodium acetate), an citrate (e.g., sodium citrate),saline, PBS, and sucrose. In certain embodiments, the pharmaceuticalcomposition of the disclosure has a pH value between about 7 and 8(e.g., 6.8 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 or 8.0,or between 7.5 and 8 or between 7 and 7.8). For example, apharmaceutical composition of the disclosure comprises a nanoparticlecomposition disclosed herein, Tris, saline and sucrose, and has a pH ofabout 7.5-8, which is suitable for storage and/or shipment at, forexample, about −20° C. For example, a pharmaceutical composition of thedisclosure comprises a nanoparticle composition disclosed herein and PBSand has a pH of about 7-7.8, suitable for storage and/or shipment at,for example, about 4° C. or lower. “Stability,” “stabilized,” and“stable” in the context of the present disclosure refers to theresistance of nanoparticle compositions and/or pharmaceuticalcompositions disclosed herein to chemical or physical changes (e.g.,degradation, particle size change, aggregation, change in encapsulation,etc.) under given manufacturing, preparation, transportation, storageand/or in-use conditions, e.g., when stress is applied such as shearforce, freeze/thaw stress, etc.

Nanoparticle compositions and/or pharmaceutical compositions includingone or more nanoparticle compositions may be administered to any patientor subject, including those patients or subjects that may benefit from atherapeutic effect provided by the delivery of a therapeutic and/orprophylactic to one or more particular cells, tissues, organs, orsystems or groups thereof, such as the renal system. Although thedescriptions provided herein of nanoparticle compositions andpharmaceutical compositions including nanoparticle compositions areprincipally directed to compositions which are suitable foradministration to humans, it will be understood by the skilled artisanthat such compositions are generally suitable for administration to anyother mammal. Modification of compositions suitable for administrationto humans in order to render the compositions suitable foradministration to various animals is well understood, and the ordinarilyskilled veterinary pharmacologist can design and/or perform suchmodification with merely ordinary, if any, experimentation. Subjects towhich administration of the compositions is contemplated include, butare not limited to, humans, other primates, and other mammals, includingcommercially relevant mammals such as cattle, pigs, hoses, sheep, cats,dogs, mice, and/or rats.

A pharmaceutical composition including one or more nanoparticlecompositions may be prepared by any method known or hereafter developedin the art of pharmacology. In general, such preparatory methods includebringing the active ingredient into association with an excipient and/orone or more other accessory ingredients, and then, if desirable ornecessary, dividing, shaping, and/or packaging the product into adesired single- or multi-dose unit.

A pharmaceutical composition in accordance with the present disclosuremay be prepared, packaged, and/or sold in bulk, as a single unit dose,and/or as a plurality of single unit doses. As used herein, a “unitdose” is discrete amount of the pharmaceutical composition comprising apredetermined amount of the active ingredient (e.g., nanoparticlecomposition). The amount of the active ingredient is generally equal tothe dosage of the active ingredient which would be administered to asubject and/or a convenient fraction of such a dosage such as, forexample, one-half or one-third of such a dosage.

Pharmaceutical compositions may be prepared in a variety of formssuitable for a variety of routes and methods of administration. Forexample, pharmaceutical compositions may be prepared in liquid dosageforms (e.g., emulsions, microemulsions, nanoemulsions, solutions,suspensions, syrups, and elixirs), injectable forms, solid dosage forms(e.g., capsules, tablets, pills, powders, and granules), dosage formsfor topical and/or transdermal administration (e.g., ointments, pastes,creams, lotions, gels, powders, solutions, sprays, inhalants, andpatches), suspensions, powders, and other forms.

Liquid dosage forms for oral and parenteral administration include, butare not limited to, pharmaceutically acceptable emulsions,microemulsions, nanoemulsions, solutions, suspensions, syrups, and/orelixirs. In addition to active ingredients, liquid dosage forms maycomprise inert diluents commonly used in the art such as, for example,water or other solvents, solubilizing agents and emulsifiers such asethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzylalcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol,dimethylformamide, oils (in particular, cottonseed, groundnut, corn,germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfurylalcohol, polyethylene glycols and fatty acid esters of sorbitan, andmixtures thereof. Besides inert diluents, oral compositions can includeadditional therapeutic and/or prophylactics, additional agents such aswetting agents, emulsifying and suspending agents, sweetening,flavoring, and/or perfuming agents. In certain embodiments forparenteral administration, compositions are mixed with solubilizingagents such as Cremophor®, alcohols, oils, modified oils, glycols,polysorbates, cyclodextrins, polymers, and/or combinations thereof.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing agents, wetting agents, and/or suspendingagents. Sterile injectable preparations may be sterile injectablesolutions, suspensions, and/or emulsions in nontoxic parenterallyacceptable diluents and/or solvents, for example, as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that may beemployed are water, Ringer's solution, U.S.P., and isotonic sodiumchloride solution. Sterile, fixed oils are conventionally employed as asolvent or suspending medium. For this purpose any bland fixed oil canbe employed including synthetic mono- or diglycerides. Fatty acids suchas oleic acid can be used in the preparation of injectables.

Injectable formulations can be sterilized, for example, by filtrationthrough a bacterial-retaining filter, and/or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium prior to use.

In order to prolong the effect of an active ingredient, it is oftendesirable to slow the absorption of the active ingredient fromsubcutaneous or intramuscular injection. This may be accomplished by theuse of a liquid suspension of crystalline or amorphous material withpoor water solubility. The rate of absorption of the drug then dependsupon its rate of dissolution which, in turn, may depend upon crystalsize and crystalline form. Alternatively, delayed absorption of aparenterally administered drug form is accomplished by dissolving orsuspending the drug in an oil vehicle. Injectable depot forms are madeby forming microencapsulated matrices of the drug in biodegradablepolymers such as polylactide-polyglycolide. Depending upon the ratio ofdrug to polymer and the nature of the particular polymer employed, therate of drug release can be controlled. Examples of other biodegradablepolymers include poly(orthoesters) and poly(anhydrides). Depotinjectable formulations are prepared by entrapping the drug in liposomesor microemulsions which are compatible with body tissues.

Compositions for rectal or vaginal administration are typicallysuppositories which can be prepared by mixing compositions with suitablenon-irritating excipients such as cocoa butter, polyethylene glycol or asuppository wax which are solid at ambient temperature but liquid atbody temperature and therefore melt in the rectum or vaginal cavity andrelease the active ingredient.

Solid dosage forms for oral administration include capsules, tablets,pills, films, powders, and granules. In such solid dosage forms, anactive ingredient is mixed with at least one inert, pharmaceuticallyacceptable excipient such as sodium citrate or dicalcium phosphateand/or fillers or extenders (e.g. starches, lactose, sucrose, glucose,mannitol, and silicic acid), binders (e.g., carboxymethylcellulose,alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia),humectants (e.g., glycerol), disintegrating agents (e.g., agar, calciumcarbonate, potato or tapioca starch, alginic acid, certain silicates,and sodium carbonate), solution retarding agents (e.g., paraffin),absorption accelerators (e.g., quaternary ammonium compounds), wettingagents (e.g., cetyl alcohol and glycerol monostearate), absorbents(e.g., kaolin and bentonite clay, silicates), and lubricants (e.g.,talc, calcium stearate, magnesium stearate, solid polyethylene glycols,sodium lauryl sulfate), and mixtures thereof. In the case of capsules,tablets and pills, the dosage form may comprise buffering agents.

Solid compositions of a similar type may be employed as fillers in softand hard-filled gelatin capsules using such excipients as lactose ormilk sugar as well as high molecular weight polyethylene glycols and thelike. Solid dosage forms of tablets, dragees, capsules, pills, andgranules can be prepared with coatings and shells such as entericcoatings and other coatings well known in the pharmaceutical formulatingart. They may optionally comprise opacifying agents and can be of acomposition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions which can beused include polymeric substances and waxes. Solid compositions of asimilar type may be employed as fillers in soft and hard-filled gelatincapsules using such excipients as lactose or milk sugar as well as highmolecular weight polyethylene glycols and the like.

Dosage forms for topical and/or transdermal administration of acomposition may include ointments, pastes, creams, lotions, gels,powders, solutions, sprays, inhalants, and/or patches. Generally, anactive ingredient is admixed under sterile conditions with apharmaceutically acceptable excipient and/or any needed preservativesand/or buffers as may be required. Additionally, the present disclosurecontemplates the use of transdermal patches, which often have the addedadvantage of providing controlled delivery of a compound to the body.Such dosage forms may be prepared, for example, by dissolving and/ordispensing the compound in the proper medium. Alternatively oradditionally, rate may be controlled by either providing a ratecontrolling membrane and/or by dispersing the compound in a polymermatrix and/or gel.

Suitable devices for use in delivering intradermal pharmaceuticalcompositions described herein include short needle devices such as thosedescribed in U.S. Pat. Nos. 4,886,499; 5,190,521; 5,328,483; 5,527,288;4,270,537; 5,015,235; 5,141,496; and 5,417,662. Intradermal compositionsmay be administered by devices which limit the effective penetrationlength of a needle into the skin, such as those described in PCTpublication WO 99/34850 and functional equivalents thereof. Jetinjection devices which deliver liquid compositions to the dermis via aliquid jet injector and/or via a needle which pierces the stratumcorneum and produces a jet which reaches the dermis are suitable. Jetinjection devices are described, for example, in U.S. Pat. Nos.5,480,381; 5,599,302; 5,334,144; 5,993,412; 5,649,912; 5,569,189;5,704,911; 5,383,851; 5,893,397; 5,466,220; 5,339,163; 5,312,335;5,503,627; 5,064,413; 5,520,639; 4,596,556; 4,790,824; 4,941,880;4,940,460; and PCT publications WO 97/37705 and WO 97/13537. Ballisticpowder/particle delivery devices which use compressed gas to acceleratevaccine in powder form through the outer layers of the skin to thedermis are suitable. Alternatively or additionally, conventionalsyringes may be used in the classical mantoux method of intradermaladministration.

Formulations suitable for topical administration include, but are notlimited to, liquid and/or semi liquid preparations such as liniments,lotions, oil in water and/or water in oil emulsions such as creams,ointments and/or pastes, and/or solutions and/or suspensions.Topically-administrable formulations may, for example, comprise fromabout 1% to about 10% (wt/wt) active ingredient, although theconcentration of active ingredient may be as high as the solubilitylimit of the active ingredient in the solvent. Formulations for topicaladministration may further comprise one or more of the additionalingredients described herein.

A pharmaceutical composition may be prepared, packaged, and/or sold in aformulation suitable for pulmonary administration via the buccal cavity.Such a formulation may comprise dry particles which comprise the activeingredient. Such compositions are conveniently in the form of drypowders for administration using a device comprising a dry powderreservoir to which a stream of propellant may be directed to dispersethe powder and/or using a self-propelling solvent/powder dispensingcontainer such as a device comprising the active ingredient dissolvedand/or suspended in a low-boiling propellant in a sealed container. Drypowder compositions may include a solid fine powder diluent such assugar and are conveniently provided in a unit dose form.

Low boiling propellants generally include liquid propellants having aboiling point of below 65° F. at atmospheric pressure. Generally thepropellant may constitute 50% to 99.9% (wt/wt) of the composition, andactive ingredient may constitute 0.1% to 20% (wt/wt) of the composition.A propellant may further comprise additional ingredients such as aliquid non-ionic and/or solid anionic surfactant and/or a solid diluent(which may have a particle size of the same order as particlescomprising the active ingredient).

Pharmaceutical compositions formulated for pulmonary delivery mayprovide an active ingredient in the form of droplets of a solutionand/or suspension. Such formulations may be prepared, packaged, and/orsold as aqueous and/or dilute alcoholic solutions and/or suspensions,optionally sterile, comprising active ingredient, and may convenientlybe administered using any nebulization and/or atomization device. Suchformulations may further comprise one or more additional ingredientsincluding, but not limited to, a flavoring agent such as saccharinsodium, a volatile oil, a buffering agent, a surface active agent,and/or a preservative such as methylhydroxybenzoate. Droplets providedby this route of administration may have an average diameter in therange from about 1 nm to about 200 nm.

Formulations described herein as being useful for pulmonary delivery areuseful for intranasal delivery of a pharmaceutical composition. Anotherformulation suitable for intranasal administration is a coarse powdercomprising the active ingredient and having an average particle fromabout 0.2 μm to 500 μm. Such a formulation is administered in the mannerin which snuff is taken, i.e. by rapid inhalation through the nasalpassage from a container of the powder held close to the nose.

Formulations suitable for nasal administration may, for example,comprise from about as little as 0.1% (wt/wt) and as much as 100%(wt/wt) of active ingredient, and may comprise one or more of theadditional ingredients described herein. A pharmaceutical compositionmay be prepared, packaged, and/or sold in a formulation suitable forbuccal administration. Such formulations may, for example, be in theform of tablets and/or lozenges made using conventional methods, andmay, for example, 0.1% to 20% (wt/wt) active ingredient, the balancecomprising an orally dissolvable and/or degradable composition and,optionally, one or more of the additional ingredients described herein.Alternately, formulations suitable for buccal administration maycomprise a powder and/or an aerosolized and/or atomized solution and/orsuspension comprising active ingredient. Such powdered, aerosolized,and/or aerosolized formulations, when dispersed, may have an averageparticle and/or droplet size in the range from about 0.1 nm to about 200nm, and may further comprise one or more of any additional ingredientsdescribed herein.

A pharmaceutical composition may be prepared, packaged, and/or sold in aformulation suitable for ophthalmic administration. Such formulationsmay, for example, be in the form of eye drops including, for example, a0.1/1.0% (wt/wt) solution and/or suspension of the active ingredient inan aqueous or oily liquid excipient. Such drops may further comprisebuffering agents, salts, and/or one or more other of any additionalingredients described herein. Other ophthalmically-administrableformulations which are useful include those which comprise the activeingredient in microcrystalline form and/or in a liposomal preparation.Ear drops and/or eye drops are contemplated as being within the scope ofthis present disclosure.

Methods of Producing Polypeptides in Cells

The present disclosure provides methods of producing a polypeptide ofinterest in a mammalian cell. Methods of producing polypeptides involvecontacting a cell with a nanoparticle composition including an mRNAencoding the polypeptide of interest. Upon contacting the cell with thenanoparticle composition, the mRNA may be taken up and translated in thecell to produce the polypeptide of interest.

In general, the step of contacting a mammalian cell with a nanoparticlecomposition including an mRNA encoding a polypeptide of interest may beperformed in vivo, ex vivo, in culture, or in vitro. The amount ofnanoparticle composition contacted with a cell, and/or the amount ofmRNA therein, may depend on the type of cell or tissue being contacted,the means of administration, the physiochemical characteristics of thenanoparticle composition and the mRNA (e.g., size, charge, and chemicalcomposition) therein, and other factors. In general, an effective amountof the nanoparticle composition will allow for efficient polypeptideproduction in the cell. Metrics for efficiency may include polypeptidetranslation (indicated by polypeptide expression), level of mRNAdegradation, and immune response indicators.

The step of contacting a nanoparticle composition including an mRNA witha cell may involve or cause transfection. A phospholipid including inthe lipid component of a nanoparticle composition may facilitatetransfection and/or increase transfection efficiency, for example, byinteracting and/or fusing with a cellular or intracellular membrane.Transfection may allow for the translation of the mRNA within the cell.

In some embodiments, the nanoparticle compositions described herein maybe used therapeutically. For example, an mRNA included in a nanoparticlecomposition may encode a therapeutic polypeptide (e.g., in atranslatable region) and produce the therapeutic polypeptide uponcontacting and/or entry (e.g., transfection) into a cell. In otherembodiments, an mRNA included in a nanoparticle composition may encode apolypeptide that may improve or increase the immunity of a subject. Forexample, an mRNA may encode a granulocyte-colony stimulating factor ortrastuzumab.

In certain embodiments, an mRNA included in a nanoparticle compositionmay encode a recombinant polypeptide that may replace one or morepolypeptides that may be substantially absent in a cell contacted withthe nanoparticle composition. The one or more substantially absentpolypeptides may be lacking due to a genetic mutation of the encodinggene or a regulatory pathway thereof. Alternatively, a recombinantpolypeptide produced by translation of the mRNA may antagonize theactivity of an endogenous protein present in, on the surface of, orsecreted from the cell. An antagonistic recombinant polypeptide may bedesirable to combat deleterious effects caused by activities of theendogenous protein, such as altered activities or localization caused bymutation. In another alternative, a recombinant polypeptide produced bytranslation of the mRNA may indirectly or directly antagonize theactivity of a biological moiety present in, on the surface of, orsecreted from the cell. Antagonized biological moieties may include, butare not limited to, lipids (e.g., cholesterol), lipoproteins (e.g., lowdensity lipoprotein), nucleic acids, carbohydrates, and small moleculetoxins. Recombinant polypeptides produced by translation of the mRNA maybe engineered for localization within the cell, such as within aspecific compartment such as the nucleus, or may be engineered forsecretion from the cell or for translocation to the plasma membrane ofthe cell.

In some embodiments, contacting a cell with a nanoparticle compositionincluding an mRNA may reduce the innate immune response of a cell to anexogenous nucleic acid. A cell may be contacted with a firstnanoparticle composition including a first amount of a first exogenousmRNA including a translatable region and the level of the innate immuneresponse of the cell to the first exogenous mRNA may be determined.Subsequently, the cell may be contacted with a second compositionincluding a second amount of the first exogenous mRNA, the second amountbeing a lesser amount of the first exogenous mRNA compared to the firstamount. Alternatively, the second composition may include a first amountof a second exogenous mRNA that is different from the first exogenousmRNA. The steps of contacting the cell with the first and secondcompositions may be repeated one or more times. Additionally, efficiencyof polypeptide production (e.g., translation) in the cell may beoptionally determined, and the cell may be re-contacted with the firstand/or second composition repeatedly until a target protein productionefficiency is achieved.

Methods of Delivering Therapeutic Agents to Cells and Organs

The present disclosure provides methods of delivering a therapeuticand/or prophylactic to a mammalian cell or organ. Delivery of atherapeutic and/or prophylactic to a cell involves administering ananoparticle composition including the therapeutic and/or prophylacticto a subject, where administration of the composition involvescontacting the cell with the composition. For example, a protein,cytotoxic agent, radioactive ion, chemotherapeutic agent, or nucleicacid (such as an RNA, e.g., mRNA) may be delivered to a cell or organ.In the instance that a therapeutic and/or prophylactic is an mRNA, uponcontacting a cell with the nanoparticle composition, a translatable mRNAmay be translated in the cell to produce a polypeptide of interest.However, mRNAs that are substantially not translatable may also bedelivered to cells. Substantially non-translatable mRNAs may be usefulas vaccines and/or may sequester translational components of a cell toreduce expression of other species in the cell.

In some embodiments, a nanoparticle composition may target a particulartype or class of cells (e.g., cells of a particular organ or systemthereof). For example, a nanoparticle composition including atherapeutic and/or prophylactic of interest may be specificallydelivered to a mammalian liver, kidney, spleen, femur, or lung. Specificdelivery to a particular class of cells, an organ, or a system or groupthereof implies that a higher proportion of nanoparticle compositionsincluding a therapeutic and/or prophylactic are delivered to thedestination (e.g., tissue) of interest relative to other destinations,e.g., upon administration of a nanoparticle composition to a mammal. Insome embodiments, specific delivery may result in a greater than 2 fold,5 fold, 10 fold, 15 fold, or 20 fold increase in the amount oftherapeutic and/or prophylactic per 1 g of tissue of the targeteddestination (e.g., tissue of interest, such as a liver) as compared toanother destination (e.g., the spleen). In some embodiments, the tissueof interest is selected from the group consisting of a liver, kidney, alung, a spleen, a femur, an ocular tissue (e.g., via intraocular,subretinal, or intravitreal injection), vascular endothelium in vessels(e.g., intra-coronary or intra-femoral) or kidney, and tumor tissue(e.g., via intratumoral injection).

As another example of targeted or specific delivery, an mRNA thatencodes a protein-binding partner (e.g., an antibody or functionalfragment thereof, a scaffold protein, or a peptide) or a receptor on acell surface may be included in a nanoparticle composition. An mRNA mayadditionally or instead be used to direct the synthesis andextracellular localization of lipids, carbohydrates, or other biologicalmoieties. Alternatively, other therapeutic and/or prophylactics orelements (e.g., lipids or ligands) of a nanoparticle composition may beselected based on their affinity for particular receptors (e.g., lowdensity lipoprotein receptors) such that a nanoparticle composition maymore readily interact with a target cell population including thereceptors. For example, ligands may include, but are not limited to,members of a specific binding pair, antibodies, monoclonal antibodies,Fv fragments, single chain Fv (scFv) fragments, Fab′ fragments, F(ab′)2fragments, single domain antibodies, camelized antibodies and fragmentsthereof, humanized antibodies and fragments thereof, and multivalentversions thereof; multivalent binding reagents including mono- orbi-specific antibodies such as disulfide stabilized Fv fragments, scFvtandems, diabodies, tribodies, or tetrabodies; and aptamers, receptors,and fusion proteins.

In some embodiments, a ligand may be a surface-bound antibody, which canpermit tuning of cell targeting specificity. This is especially usefulsince highly specific antibodies can be raised against an epitope ofinterest for the desired targeting site. In one embodiment, multipleantibodies are expressed on the surface of a cell, and each antibody canhave a different specificity for a desired target. Such approaches canincrease the avidity and specificity of targeting interactions.

A ligand can be selected, e.g., by a person skilled in the biologicalarts, based on the desired localization or function of the cell. Forexample an estrogen receptor ligand, such as tamoxifen, can target cellsto estrogen-dependent breast cancer cells that have an increased numberof estrogen receptors on the cell surface. Other non-limiting examplesof ligand/receptor interactions include CCR1 (e.g., for treatment ofinflamed joint tissues or brain in rheumatoid arthritis, and/or multiplesclerosis), CCR7, CCR8 (e.g., targeting to lymph node tissue), CCR6,CCR9, CCR10 (e.g., to target to intestinal tissue), CCR4, CCR10 (e.g.,for targeting to skin), CXCR4 (e.g., for general enhancedtransmigration), HCELL (e.g., for treatment of inflammation andinflammatory disorders, bone marrow), Alpha4beta7 (e.g., for intestinalmucosa targeting), and VLA-4NCAM-1 (e.g., targeting to endothelium). Ingeneral, any receptor involved in targeting (e.g., cancer metastasis)can be harnessed for use in the methods and compositions describedherein.

Targeted cells may include, but are not limited to, hepatocytes,epithelial cells, hematopoietic cells, epithelial cells, endothelialcells, lung cells, bone cells, stem cells, mesenchymal cells, neuralcells, cardiac cells, adipocytes, vascular smooth muscle cells,cardiomyocytes, skeletal muscle cells, beta cells, pituitary cells,synovial lining cells, ovarian cells, testicular cells, fibroblasts, Bcells, T cells, reticulocytes, leukocytes, granulocytes, and tumorcells.

In some embodiments, a nanoparticle composition may target hepatocytes.Apolipoprotiens such as apolipoprotein E (apoE) have been shown toassociate with neutral or near neutral lipid-containing nanoparticlecompositions in the body, and are known to associate with receptors suchas low-density lipoprotein receptors (LDLRs) found on the surface ofhepatocytes. Thus, a nanoparticle composition including a lipidcomponent with a neutral or near neutral charge that is administered toa subject may acquire apoE in a subject's body and may subsequentlydeliver a therapeutic and/or prophylactic (e.g., an RNA) to hepatocytesincluding LDLRs in a targeted manner.

Methods of Treating Diseases and Disorders

Nanoparticle compositions may be useful for treating a disease,disorder, or condition. In particular, such compositions may be usefulin treating a disease, disorder, or condition characterized by missingor aberrant protein or polypeptide activity. For example, a nanoparticlecomposition comprising an mRNA encoding a missing or aberrantpolypeptide may be administered or delivered to a cell. Subsequenttranslation of the mRNA may produce the polypeptide, thereby reducing oreliminating an issue caused by the absence of or aberrant activitycaused by the polypeptide. Because translation may occur rapidly, themethods and compositions may be useful in the treatment of acutediseases, disorders, or conditions such as sepsis, stroke, andmyocardial infarction. A therapeutic and/or prophylactic included in ananoparticle composition may also be capable of altering the rate oftranscription of a given species, thereby affecting gene expression.

Diseases, disorders, and/or conditions characterized by dysfunctional oraberrant protein or polypeptide activity for which a composition may beadministered include, but are not limited to, rare diseases, infectiousdiseases (as both vaccines and therapeutics), cancer and proliferativediseases, genetic diseases (e.g., cystic fibrosis), autoimmune diseases,diabetes, neurodegenerative diseases, cardio- and reno-vasculardiseases, and metabolic diseases. Multiple diseases, disorders, and/orconditions may be characterized by missing (or substantially diminishedsuch that proper protein function does not occur) protein activity. Suchproteins may not be present, or they may be essentially non-functional.A specific example of a dysfunctional protein is the missense mutationvariants of the cystic fibrosis transmembrane conductance regulator(CFTR) gene, which produce a dysfunctional protein variant of CFTRprotein, which causes cystic fibrosis. The present disclosure provides amethod for treating such diseases, disorders, and/or conditions in asubject by administering a nanoparticle composition including an RNA anda lipid component including a lipid according to Formula (I), aphospholipid (optionally unsaturated), a PEG lipid, and a structurallipid, wherein the RNA may be an mRNA encoding a polypeptide thatantagonizes or otherwise overcomes an aberrant protein activity presentin the cell of the subject.

The disclosure provides methods involving administering nanoparticlecompositions including one or more therapeutic and/or prophylacticagents and pharmaceutical compositions including the same. The termstherapeutic and prophylactic can be used interchangeably herein withrespect to features and embodiments of the present disclosure.Therapeutic compositions, or imaging, diagnostic, or prophylacticcompositions thereof, may be administered to a subject using anyreasonable amount and any route of administration effective forpreventing, treating, diagnosing, or imaging a disease, disorder, and/orcondition and/or any other purpose. The specific amount administered toa given subject may vary depending on the species, age, and generalcondition of the subject; the purpose of the administration; theparticular composition; the mode of administration; and the like.Compositions in accordance with the present disclosure may be formulatedin dosage unit form for ease of administration and uniformity of dosage.It will be understood, however, that the total daily usage of acomposition of the present disclosure will be decided by an attendingphysician within the scope of sound medical judgment. The specifictherapeutically effective, prophylactically effective, or otherwiseappropriate dose level (e.g., for imaging) for any particular patientwill depend upon a variety of factors including the severity andidentify of a disorder being treated, if any; the one or moretherapeutic and/or prophylactics employed; the specific compositionemployed; the age, body weight, general health, sex, and diet of thepatient; the time of administration, route of administration, and rateof excretion of the specific pharmaceutical composition employed; theduration of the treatment; drugs used in combination or coincidentalwith the specific pharmaceutical composition employed; and like factorswell known in the medical arts.

A nanoparticle composition including one or more therapeutic and/orprophylactics may be administered by any route. In some embodiments,compositions, including prophylactic, diagnostic, or imagingcompositions including one or more nanoparticle compositions describedherein, are administered by one or more of a variety of routes,including oral, intravenous, intramuscular, intra-arterial,intramedullary, intrathecal, subcutaneous, intraventricular, trans- orintra-dermal, interdermal, rectal, intravaginal, intraperitoneal,intraocular, subretinal, intravitreal, topical (e.g. by powders,ointments, creams, gels, lotions, and/or drops), mucosal, nasal, buccal,enteral, vitreal, intratumoral, sublingual, intranasal; by intratrachealinstillation, bronchial instillation, and/or inhalation; as an oralspray and/or powder, nasal spray, and/or aerosol, and/or through aportal vein catheter. In some embodiments, a composition may beadministered intravenously, intramuscularly, intradermally,intra-arterially, intratumorally, subcutaneously, intraocularly,subretinally, intravitreally, or by inhalation. However, the presentdisclosure encompasses the delivery or administration of compositionsdescribed herein by any appropriate route taking into considerationlikely advances in the sciences of drug delivery. In general, the mostappropriate route of administration will depend upon a variety offactors including the nature of the nanoparticle composition includingone or more therapeutic and/or prophylactics (e.g., its stability invarious bodily environments such as the bloodstream and gastrointestinaltract), the condition of the patient (e.g., whether the patient is ableto tolerate particular routes of administration), etc.

In certain embodiments, compositions in accordance with the presentdisclosure may be administered at dosage levels sufficient to deliverfrom about 0.0001 mg/kg to about 10 mg/kg, from about 0.001 mg/kg toabout 10 mg/kg, from about 0.005 mg/kg to about 10 mg/kg, from about0.01 mg/kg to about 10 mg/kg, from about 0.05 mg/kg to about 10 mg/kg,from about 0.1 mg/kg to about 10 mg/kg, from about 1 mg/kg to about 10mg/kg, from about 2 mg/kg to about 10 mg/kg, from about 5 mg/kg to about10 mg/kg, from about 0.0001 mg/kg to about 5 mg/kg, from about 0.001mg/kg to about 5 mg/kg, from about 0.005 mg/kg to about 5 mg/kg, fromabout 0.01 mg/kg to about 5 mg/kg, from about 0.05 mg/kg to about 5mg/kg, from about 0.1 mg/kg to about 5 mg/kg, from about 1 mg/kg toabout 5 mg/kg, from about 2 mg/kg to about 5 mg/kg, from about 0.0001mg/kg to about 2.5 mg/kg, from about 0.001 mg/kg to about 2.5 mg/kg,from about 0.005 mg/kg to about 2.5 mg/kg, from about 0.01 mg/kg toabout 2.5 mg/kg, from about 0.05 mg/kg to about 2.5 mg/kg, from about0.1 mg/kg to about 2.5 mg/kg, from about 1 mg/kg to about 2.5 mg/kg,from about 2 mg/kg to about 2.5 mg/kg, from about 0.0001 mg/kg to about1 mg/kg, from about 0.001 mg/kg to about 1 mg/kg, from about 0.005 mg/kgto about 1 mg/kg, from about 0.01 mg/kg to about 1 mg/kg, from about0.05 mg/kg to about 1 mg/kg, from about 0.1 mg/kg to about 1 mg/kg, fromabout 0.0001 mg/kg to about 0.25 mg/kg, from about 0.001 mg/kg to about0.25 mg/kg, from about 0.005 mg/kg to about 0.25 mg/kg, from about 0.01mg/kg to about 0.25 mg/kg, from about 0.05 mg/kg to about 0.25 mg/kg, orfrom about 0.1 mg/kg to about 0.25 mg/kg of a therapeutic and/orprophylactic (e.g., an mRNA) in a given dose, where a dose of 1 mg/kg(mpk) provides 1 mg of a therapeutic and/or prophylactic per 1 kg ofsubject body weight. In some embodiments, a dose of about 0.001 mg/kg toabout 10 mg/kg of a therapeutic and/or prophylactic (e.g., mRNA) of ananoparticle composition may be administered. In other embodiments, adose of about 0.005 mg/kg to about 2.5 mg/kg of a therapeutic and/orprophylactic may be administered. In certain embodiments, a dose ofabout 0.1 mg/kg to about 1 mg/kg may be administered. In otherembodiments, a dose of about 0.05 mg/kg to about 0.25 mg/kg may beadministered. A dose may be administered one or more times per day, inthe same or a different amount, to obtain a desired level of mRNAexpression and/or therapeutic, diagnostic, prophylactic, or imagingeffect. The desired dosage may be delivered, for example, three times aday, two times a day, once a day, every other day, every third day,every week, every two weeks, every three weeks, or every four weeks. Incertain embodiments, the desired dosage may be delivered using multipleadministrations (e.g., two, three, four, five, six, seven, eight, nine,ten, eleven, twelve, thirteen, fourteen, or more administrations). Insome embodiments, a single dose may be administered, for example, priorto or after a surgical procedure or in the instance of an acute disease,disorder, or condition.

Nanoparticle compositions including one or more therapeutic and/orprophylactics may be used in combination with one or more othertherapeutic, prophylactic, diagnostic, or imaging agents. By “incombination with,” it is not intended to imply that the agents must beadministered at the same time and/or formulated for delivery together,although these methods of delivery are within the scope of the presentdisclosure. For example, one or more nanoparticle compositions includingone or more different therapeutic and/or prophylactics may beadministered in combination. Compositions can be administeredconcurrently with, prior to, or subsequent to, one or more other desiredtherapeutics or medical procedures. In general, each agent will beadministered at a dose and/or on a time schedule determined for thatagent. In some embodiments, the present disclosure encompasses thedelivery of compositions, or imaging, diagnostic, or prophylacticcompositions thereof in combination with agents that improve theirbioavailability, reduce and/or modify their metabolism, inhibit theirexcretion, and/or modify their distribution within the body.

It will further be appreciated that therapeutically, prophylactically,diagnostically, or imaging active agents utilized in combination may beadministered together in a single composition or administered separatelyin different compositions. In general, it is expected that agentsutilized in combination will be utilized at levels that do not exceedthe levels at which they are utilized individually. In some embodiments,the levels utilized in combination may be lower than those utilizedindividually.

The particular combination of therapies (therapeutics or procedures) toemploy in a combination regimen will take into account compatibility ofthe desired therapeutics and/or procedures and the desired therapeuticeffect to be achieved. It will also be appreciated that the therapiesemployed may achieve a desired effect for the same disorder (forexample, a composition useful for treating cancer may be administeredconcurrently with a chemotherapeutic agent), or they may achievedifferent effects (e.g., control of any adverse effects, such asinfusion related reactions).

A nanoparticle composition may be used in combination with an agent toincrease the effectiveness and/or therapeutic window of the composition.Such an agent may be, for example, an anti-inflammatory compound, asteroid (e.g., a corticosteroid), a statin, an estradiol, a BTKinhibitor, an S1P1 agonist, a glucocorticoid receptor modulator (GRM),or an anti-histamine. In some embodiments, a nanoparticle compositionmay be used in combination with dexamethasone, methotrexate,acetaminophen, an H1 receptor blocker, or an H2 receptor blocker. Insome embodiments, a method of treating a subject in need thereof or ofdelivering a therapeutic and/or prophylactic to a subject (e.g., amammal) may involve pre-treating the subject with one or more agentsprior to administering a nanoparticle composition. For example, asubject may be pre-treated with a useful amount (e.g., 10 mg, 20 mg, 30mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, or any otheruseful amount) of dexamethasone, methotrexate, acetaminophen, an H1receptor blocker, or an H2 receptor blocker. Pre-treatment may occur 24or fewer hours (e.g., 24 hours, 20 hours, 16 hours, 12 hours, 8 hours, 4hours, 2 hours, 1 hour, 50 minutes, 40 minutes, 30 minutes, 20 minutes,or 10 minutes) before administration of the nanoparticle composition andmay occur one, two, or more times in, for example, increasing dosageamounts.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments in accordance with the disclosure described herein. Thescope of the present disclosure is not intended to be limited to theabove Description, but rather is as set forth in the appended claims.

In the claims, articles such as “a,” “an,” and “the” may mean one ormore than one unless indicated to the contrary or otherwise evident fromthe context. Claims or descriptions that include “or” between one ormore members of a group are considered satisfied if one, more than one,or all of the group members are present in, employed in, or otherwiserelevant to a given product or process unless indicated to the contraryor otherwise evident from the context. The disclosure includesembodiments in which exactly one member of the group is present in,employed in, or otherwise relevant to a given product or process. Thedisclosure includes embodiments in which more than one, or all, of thegroup members are present in, employed in, or otherwise relevant to agiven product or process.

It is also noted that the term “comprising” is intended to be open andpermits but does not require the inclusion of additional elements orsteps. When the term “comprising” is used herein, the terms “consistingessentially of” and “consisting of” are thus also encompassed anddisclosed. Throughout the description, where compositions are describedas having, including, or comprising specific components, it iscontemplated that compositions also consist essentially of, or consistof, the recited components. Similarly, where methods or processes aredescribed as having, including, or comprising specific process steps,the processes also consist essentially of, or consist of, the recitedprocessing steps. Further, it should be understood that the order ofsteps or order for performing certain actions is immaterial so long asthe invention remains operable. Moreover, two or more steps or actionscan be conducted simultaneously.

Where ranges are given, endpoints are included. Furthermore, it is to beunderstood that unless otherwise indicated or otherwise evident from thecontext and understanding of one of ordinary skill in the art, valuesthat are expressed as ranges can assume any specific value or sub-rangewithin the stated ranges in different embodiments of the disclosure, tothe tenth of the unit of the lower limit of the range, unless thecontext clearly dictates otherwise.

The synthetic processes of the disclosure can tolerate a wide variety offunctional groups, therefore various substituted starting materials canbe used. The processes generally provide the desired final compound ator near the end of the overall process, although it may be desirable incertain instances to further convert the compound to a pharmaceuticallyacceptable salt thereof.

Compounds of the present disclosure can be prepared in a variety of waysusing commercially available starting materials, compounds known in theliterature, or from readily prepared intermediates, by employingstandard synthetic methods and procedures either known to those skilledin the art, or which will be apparent to the skilled artisan in light ofthe teachings herein. Standard synthetic methods and procedures for thepreparation of organic molecules and functional group transformationsand manipulations can be obtained from the relevant scientificliterature or from standard textbooks in the field. Although not limitedto any one or several sources, classic texts such as Smith, M. B.,March, J., March's Advanced Organic Chemistry. Reactions, Mechanisms,and Structure, 5^(th) edition, John Wiley & Sons: New York, 2001;Greene, T. W., Wuts, P. G. M., Protective Groups in Organic Synthesis,3^(rd) edition, John Wiley & Sons: New York, 1999; R. Larock,Comprehensive Organic Transformations, VCH Publishers (1989); L. Fieserand M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis, JohnWiley and Sons (1994); and L. Paquette, ed., Encyclopedia of Reagentsfor Organic Synthesis, John Wiley and Sons (1995), incorporated byreference herein, are useful and recognized reference textbooks oforganic synthesis known to those in the art. The following descriptionsof synthetic methods are designed to illustrate, but not to limit,general procedures for the preparation of compounds of the presentdisclosure.

The compounds of this disclosure having any of the formulae describedherein may be prepared according to the procedures illustrated inSchemes 1 and 2 below, from commercially available starting materials orstarting materials which can be prepared using literature procedures.The variables in the schemes (e.g., R₁, R₂, and R₃ etc. are as definedherein). One of ordinary skill in the art will note that, during thereaction sequences and synthetic schemes described herein, the order ofcertain steps may be changed, such as the introduction and removal ofprotecting groups.

One of ordinary skill in the art will recognize that certain groups mayrequire protection from the reaction conditions via the use ofprotecting groups. Protecting groups may also be used to differentiatesimilar functional groups in molecules. A list of protecting groups andhow to introduce and remove these groups can be found in Greene, T. W.,Wuts, P. G. M., Protective Groups in Organic Synthesis, 3^(rd) edition,John Wiley & Sons: New York, 1999.

Preferred protecting groups include, but are not limited to:

For a hydroxyl moiety: TBS, benzyl, THP, Ac;

For carboxylic acids: benzyl ester, methyl ester, ethyl ester, allylester;

For amines: Fmoc, Cbz, BOC, DMB, Ac, Bn, Tr, Ts, trifluoroacetyl,phthalimide, benzylideneamine;

For diols: Ac (x2) TBS (x2), or when taken together acetonides;

For thiols: Ac;

For benzimidazoles: SEM, benzyl, PMB, DMB;

For aldehydes: di-alkyl acetals such as dimethoxy acetal or diethylacetyl.

In the reaction schemes described herein, multiple stereoisomers may beproduced. When no particular stereoisomer is indicated, it is understoodto mean all possible stereoisomers that could be produced from thereaction. A person of ordinary skill in the art will recognize that thereactions can be optimized to give one isomer preferentially, or newschemes may be devised to produce a single isomer. If mixtures areproduced, techniques such as preparative thin layer chromatography,preparative HPLC, preparative chiral HPLC, or preparative SFC may beused to separate the isomers.

As illustrated in Scheme 1 above, 8-bromooctanoic acid reacts with analcohol a1 (e.g., heptadecan-9-ol) to afford an ester b1 (e.g.,heptadecan-9-yl 8-bromooctanoate). Step 1 can take place in an organicsolvent (e.g., dichloromethane) in the presence of, e.g.,N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride,N,N-diisopropylethylamine and DMAP. Step 1 can take place at roomtemperature for 18 h. Next, ester b1 reacts with 2-aminoethan-1-ol toafford amine c1 (e.g., heptadecan-9-yl8-((2-hydroxyethyl)amino)octanoate). Step 2 can take place in ethanolat, e.g., a temperature of about 60° C. Then amine c1 reacts with anbromoalkyl R₁—Br (e.g., 1-bromotetradecane) to afford compound d1 (e.g.,heptadecan-9-yl 8-((2-hydroxyethyl)(tetradecyl)amino)octanoate). Step 3can take place in ethanol in the presence of N,N-diisopropylethylamine.

As illustrated in Scheme 2 above, an acid a2 (t is an integer between 1and 7; e.g., 8-bromooctanoic acid) reacts with an alcohol b2 (e.g.,nonan-1-ol) to afford an ester c2 (e.g., nonyl-8-bromooctanoate). Step 1can take place in an organic solvent (e.g., dichloromethane) in thepresence of, e.g., N-(3-dimethylaminopropyl)-N′-ethylcarbodiimidehydrochloride, N,N-diisopropylethylamine and DMAP. Alcohol e2 (e.g.,heptadecan-9-ol) can be obtained from reacting aldehyde d2 (e.g.,nonanal) with a Grignard reagent R₃—MgX (e.g., n-C₈H₁₇MgBr) via Step 2.Next, 8-bromooctanoic acid reacts with an alcohol e2 (e.g.,heptadecan-9-ol) to afford an ester f2 (e.g., heptadecan-9-yl8-bromooctanoate). Step 3 can take place in an organic solvent (e.g.,dichloromethane) in the presence of, e.g.,N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride,N,N-diisopropylethylamine and DMAP. Next, ester f2 reacts with2-aminoethan-1-ol to afford amine g2 (e.g., heptadecan-9-yl8-((2-hydroxyethyl)amino)octanoate). Step 4 can take place in ethanol inthe presence of i-Pr₂EtN. Then amine g2 reacts with ester c2 (e.g.,nonyl-8-bromooctanoate) to afford compound h2 (e.g., heptadecan-9-yl8-((2-hydroxyethyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate). Step 5 cantake place in an organic solvent (e.g., a mixture of CPME and MeCN), inthe presence of a base (such as an inorganic base (e.g., K₂CO₃) ornon-nucleophilic organic base (e.g., i-Pr₂EtN)) and a catalyst (e.g., aniodide such as KI or NaI) at, e.g., an elevated temperature (such as atabout 70-90° C., e.g., about 80° C.).

A person of ordinary skill in the art will recognize that in the aboveschemes the order of certain steps may be interchangeable.

In certain aspects, the disclosure also includes methods of synthesizinga compound of any of Formulae (I), (IA), (II), (IIa), (IIb), (IIc),(IId) or (IIe) and intermediate(s) for synthesizing the compound.

In some embodiments, the method of synthesizing a compound of Formula(I) includes reacting a compound of Formula (X2):

with R₁—Br to afford the compound of Formula (I), wherein each variablesare as defined herein. For example, m is 5, 6, 7, 8, or 9, preferably 5,7, or 9. For example, each of R₅, R₆, and R₇ is H. For example, M is—C(O)O— or —OC(O)—. For example, R₄ is unsubstituted C₁₋₃ alkyl, or—(CH₂)_(n)Q, in which n is 2, 3, or 4 and Q is OH, —NHC(S)N(R)₂,—NHC(O)N(R)₂, —N(R)C(O)R, or —N(R)S(O)₂R. For example, the reaction ofthe compound of Formula (X2) with R₁—Br takes place in the presence of abase (such as an inorganic base (e.g., K₂CO₃) or non-nucleophilicorganic base (e.g., i-Pr₂EtN)). For example, the reaction takes place inthe presence of an inorganic base (e.g., K₂CO₃) and a catalyst (e.g., aniodide such as KI or NaI). For example, the reaction takes place at anelevated temperature, e.g., about 50-100° C., 70-90° C., or about 80°C.).

The method may also include reacting a compound of Formula (X1):

with R₄NH₂ to afford a compound of Formula (X2), wherein each variablesare as defined herein.

In some embodiments, the intermediate(s) include those having any ofFormulae (X1) and (X2):

wherein each variables are as defined herein. For example, theintermediate includes heptadecan-9-yl 8-bromooctanoate, andheptadecan-9-yl 8-((2-hydroxyethyl)amino)octanoate, and morphic formsthereof (e.g., a crystalline form).

In addition, it is to be understood that any particular embodiment ofthe present disclosure that falls within the prior art may be explicitlyexcluded from any one or more of the claims. Since such embodiments aredeemed to be known to one of ordinary skill in the art, they may beexcluded even if the exclusion is not set forth explicitly herein.

All cited sources, for example, references, publications, databases,database entries, and art cited herein, are incorporated into thisapplication by reference, even if not expressly stated in the citation.In case of conflicting statements of a cited source and the instantapplication, the statement in the instant application shall control.

EXAMPLES Example 1: Synthesis of Compounds According to Formula (I),(IA), (II), (IIa), (IIb), (IIc), (IId) or (IIe) A. GeneralConsiderations

All solvents and reagents used were obtained commercially and used assuch unless noted otherwise. ¹H NMR spectra were recorded in CDCl₃, at300 K using a Bruker Ultrashield 300 MHz instrument. Chemical shifts arereported as parts per million (ppm) relative to TMS (0.00) for ¹H.Silica gel chromatographies were performed on ISCO CombiFlash Rf+ LumenInstruments using ISCO RediSep Rf Gold Flash Cartridges (particle size:20-40 microns). Reverse phase chromatographies were performed on ISCOCombiFlash Rf+ Lumen Instruments using RediSep Rf Gold C18 HighPerformance columns. All final compounds were determined to be greaterthan 85% pure via analysis by reverse phase UPLC-MS (retention times,RT, in minutes) using Waters Acquity UPLC instrument with DAD and ELSDand a ZORBAX Rapid Resolution High Definition (RRHD) SB-C18 LC column,2.1 mm, 50 mm, 1.8 μm, and a gradient of 65 to 100% acetonitrile inwater with 0.1% TFA over 5 minutes at 1.2 mL/min. Injection volume was 5μL and the column temperature was 80° C. Detection was based onelectrospray ionization (ESI) in positive mode using Waters SQD massspectrometer (Milford, Mass., USA) and evaporative light scatteringdetector.

The procedures described below are useful in the synthesis of Compounds1-147.

The following abbreviations are employed herein:

THF: Tetrahydrofuran

MeCN: Acetonitrile

LAH: Lithium Aluminum Hydride

DCM: Dichloromethane

DMAP: 4-Dimethylaminopyridine

LDA: Lithium Diisopropylamide

rt: Room Temperature

DME: 1,2-Dimethoxyethane

n-BuLi: n-Butyllithium

CPME: Cyclopentyl methyl ether

i-Pr₂EtN: N,N-Diisopropylethylamine

B. Compound 2: Heptadecan-9-yl8-((2-hydroxyethyl)(tetradecyl)amino)octanoate Representative Procedure1

Heptadecan-9-yl 8-bromooctanoate (Method A)

To a solution of 8-bromooctanoic acid (1.04 g, 4.6 mmol) andheptadecan-9-ol (1.5 g, 5.8 mmol) in dichloromethane (20 mL) was addedN-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (1.1 g, 5.8mmol), N,N-diisopropylethylamine (3.3 mL, 18.7 mmol) and DMAP (114 mg,0.9 mmol). The reaction was allowed to stir at rt for 18 h. The reactionwas diluted with dichloromethane and washed with saturated sodiumbicarbonate. The organic layer was separated and washed with brine, anddried over MgSO₄. The organic layer was filtered and evaporated invacuo. The residue was purified by silica gel chromatography (0-10%ethyl acetate in hexanes) to obtain heptadecan-9-yl 8-bromooctanoate(875 mg, 1.9 mmol, 41%). ¹H NMR (300 MHz, CDCl₃) δ: ppm 4.89 (m, 1H);3.42 (m, 2H); 2.31 (m, 2H); 1.89 (m, 2H); 1.73-1.18 (br. m, 36H); 0.88(m, 6H).

Heptadecan-9-yl 8-((2-hydroxyethyl)amino)octanoate (Method B)

A solution of heptadecan-9-yl 8-bromooctanoate (3.8 g, 8.2 mmol) and2-aminoethan-1-ol (15 mL, 248 mmol) in ethanol (3 mL) was allowed tostir at 62° C. for 18 h. The reaction mixture was concentrated in vacuoand the residue was taken-up in ethyl acetate and water. The organiclayer was separated and washed with water, brine and dried over Na₂SO₄.The mixture was filtered and evaporated in vacuo. The residue waspurified by silica gel chromatography (0-100% (mixture of 1% NH₄OH, 20%MeOH in dichloromethane) in dichloromethane) to obtain heptadecan-9-yl8-((2-hydroxyethyl)amino)octanoate (3.1 g, 7 mmol, 85%). UPLC/ELSD:RT=2.67 min. MS (ES): m/z (MH⁺) 442.68 for C₂₇H₅₅NO₃

¹H NMR (300 MHz, CDCl₃) δ: ppm 4.89 (p, 1H); 3.67 (t, 2H); 2.81 (t, 2H);2.65 (t, 2H); 2.30 (t, 2H); 2.05 (br. m, 2H); 1.72-1.41 (br. m, 8H);1.40-1.20 (br. m, 30H); 0.88 (m, 6H).

Heptadecan-9-yl 8-((2-hydroxyethyl)(tetradecyl)amino)octanoate (MethodC)

A solution of heptadecan-9-yl 8-((2-hydroxyethyl)amino)octanoate (125mg, 0.28 mmol), 1-bromotetradecane (94 mg, 0.34 mmol) andN,N-diisopropylethylamine (44 mg, 0.34 mmol) in ethanol was allowed tostir at 65° C. for 18 h. The reaction was cooled to rt and solvents wereevaporated in vacuo. The residue was taken-up in ethyl acetate andsaturated sodium bicarbonate. The organic layer was separated, driedover Na₂SO₄ and evaporated in vacuo. The residue was purified by silicagel chromatography (0-100% (mixture of 1% NH₄OH, 20% MeOH indichloromethane) in dichloromethane) to obtain heptadecan-9-yl8-((2-hydroxyethyl)(tetradecyl)amino)octanoate (89 mg, 0.14 mmol, 50%).UPLC/ELSD: RT=3.61 min. MS (ES): m/z (MH⁺) 638.91 for C₄₁H₈₃NO₃. ¹H NMR(300 MHz, CDCl₃) δ: ppm 4.86 (p, 1H); 3.72-3.47 (br. m, 2H); 2.78-2.40(br. m, 5H); 2.28 (t, 2H); 1.70-1.40 (m, 10H); 1.38-1.17 (br. m, 54H);0.88 (m, 9H).

Synthesis of Intermediates Intermediate A: 2-Octyldecanoic Acid

A solution of diisopropylamine (2.92 mL, 20.8 mmol) in THF (10 mL) wascooled to −78° C. and a solution of n-BuLi (7.5 mL, 18.9 mmol, 2.5 M inhexanes) was added. The reaction was allowed to warm to 0° C. To asolution of decanoic acid (2.96 g, 17.2 mmol) and NaH (754 mg, 18.9mmol, 60% w/w) in THF (20 mL) at 0° C. was added the solution of LDA andthe mixture was allowed to stir at rt for 30 min. After this time1-iodooctane (5 g, 20.8 mmol) was added and the reaction mixture washeated at 45° C. for 6 h. The reaction was quenched with 1N HCl (10 mL).The organic layer was dried over MgSO₄, filtered and evaporated invacuo. The residue was purified by silica gel chromatography (0-20%ethyl acetate in hexanes) to yield 2-octyldecanoic acid (1.9 g, 6.6mmol, 38%). ¹H NMR (300 MHz, CDCl₃) δ: ppm 2.38 (br. m, 1H); 1.74-1.03(br. m, 28H); 0.91 (m, 6H).

Intermediate B: 7-Bromoheptyl 2-octyldecanoate

7-bromoheptyl 2-octyldecanoate was synthesized using Method A from2-octyldecanoic acid and 7-bromoheptan-1-ol. ¹H NMR (300 MHz, CDCl₃) δ:ppm 4.09 (br. m, 2H); 3.43 (br. m, 2H); 2.48-2.25 (br. m, 1H); 1.89 (br.m, 2H); 1.74-1.16 (br. m, 36H); 0.90 (m, 6H).

Intermediate C: (2-Hexylcyclopropyl)methanol

A solution of diethyl zinc (20 mL, 20 mmol, 1 M in hexanes), indichloromethane (20 mL) was allowed to cool to −40° C. for 5 min. Then asolution of diiodomethane (3.22 mL, 40 mmol) in dichloromethane (10 mL)was added dropwise. After the reaction was allowed to stir for 1 h at−40° C., a solution of trichloro-acetic acid (327 mg, 2 mmol) and DME (1mL, 9.6 mmol) in dichloromethane (10 mL) was added. The reaction wasallowed to warm to −15° C. and stir at this temperature for 1 h. Asolution of (Z)-non-2-en-1-ol (1.42 g, 10 mmol) in dichloromethane (10mL) was then added to the −15° C. solution. The reaction was then slowlyallowed to warm to rt and stir for 18 h. After this time saturated NH₄Cl(200 mL) was added and the reaction was extracted with dichloromethane(3λ), washed with brine, and dried over Na₂SO₄. The organic layer wasfiltered, evaporated in vacuo and the residue was purified by silica gelchromatography (0-50% ethyl acetate in hexanes) to yield(2-hexylcyclopropyl)methanol (1.43 g, 9.2 mmol, 92%). ¹H NMR (300 MHz,CDCl₃) δ: ppm 3.64 (m, 2H); 1.57-1.02 (m, 12H); 0.99-0.80 (m, 4H); 0.72(m, 1H), 0.00 (m, 1H).

C. Compound 1: Heptadecan-9-yl8-((2-hydroxyethyl)(octadecyl)amino)octanoate

Compound 1 was synthesized according to the general procedure andRepresentative Procedure 1 described above. UPLC/ELSD: RT=3.86 min. MS(ES): m/z (MH⁺) 694.93 for C₄₅H₉₁NO₃. ¹H NMR (300 MHz, CDCl₃) δ: ppm4.86 (m, 1H); 3.77-3.47 (br. m, 2H); 2.78-2.37 (br. m, 5H); 2.28 (t,2H); 1.73-1.40 (br. m, 10H); 1.38-1.18 (br. m, 62H); 0.88 (m, 9H).

D. Compound 3: Heptadecan-9-yl 8-((2-hydroxyethyl)(nonyl)amino)octanoate

Compound 3 was synthesized according to the general procedure andRepresentative Procedure 1 and Representative Procedure 1 describedabove. UPLC/ELSD: RT=3.36 min. MS (ES): m/z (MH⁺) 568.80 for C₃₆H₇₃NO₃.¹H NMR (300 MHz, CDCl₃) δ: ppm 4.86 (p, 1H); 3.72-3.45 (br. m, 2H);2.79-2.34 (br. m, 5H); 2.28 (t, 2H); 1.70-1.38 (m, 10H); 1.38-1.16 (br.m, 44H); 0.88 (m, 9H).

E. Compound 4: Heptadecan-9-yl 8-((2-hydroxyethyl)(octyl)amino)octanoate

Compound 4 was synthesized according to the general procedure andRepresentative Procedure 1 described above. UPLC/ELSD: RT=2.99 min. MS(ES): m/z (MH⁺) 554.777 for C₃₅H₇₁NO₃. ¹H NMR (300 MHz, CDCl₃) δ: ppm4.86 (p, 1H); 3.71 (br. s, 2H); 2.70 (br. s, 5H); 2.26 (t, 2H);1.48-1.59 (br. m., 10H); 1.24 (m, 42H); 0.86 (t, 9H).

F. Compound 5: Heptadecan-9-yl 8-(hexyl(2-hydroxyethyl)amino)octanoate

Compound 5 was synthesized according to the general procedure andRepresentative Procedure 1 described above. UPLC/ELSD: RT=3.10 min. MS(ES): m/z (MH⁺) 526.73 for C₃₃H₆₇NO₃. ¹H NMR (300 MHz, CDCl₃) δ: ppm4.86 (p, 1H); 3.67-3.48 (br. m, 2H); 2.74-2.39 (br. m, 5H); 2.28 (t,2H); 1.68-1.39 (br. m, 10H); 1.38-1.16 (br. m, 38H); 0.88 (m, 9H).

G. Compound 6: Heptadecan-9-yl8-((2-hydroxyethyl)((9Z,12Z)-octadeca-9,12-dien-1-yl)amino)octanoate

Compound 6 was synthesized according to the general procedure andRepresentative Procedure 1 described above. UPLC/ELSD: RT=3.77 min. MS(ES): m/z (MH⁺) 690.84 for C₄₅H₈₇NO₃. ¹H NMR (300 MHz, CDCl₃) δ: ppm5.37 (m, 4H); 4.86 (br. m, 1H); 3.53 (br. m; 2H); 2.78 (br. m, 2H); 2.58(br. m, 2H); 2.45 (br. m, 4H); 2.28 (m, 2H); 2.05 (m, 4H); 1.68-1.15(br. m, 57H); 0.89 (m, 9H).

H. Compound 7: Heptadecan-9-yl8-((3-hydroxypropyl)(nonyl)amino)octanoate

Compound 7 was synthesized according to the general procedure andRepresentative Procedure 1 described above. UPLC/ELSD: RT=3.24 min. MS(ES): m/z (MH⁺) 582.987 for C₃₇H₇₅NO₃. ¹H NMR (300 MHz, CDCl₃) δ: ppm4.84 (p, 1H); 3.76 (t, 2H); 2.42-2.66 (br. s, 5H); 2.25 (t, 2H);1.47-1.68 (br. m, 12H); 1.24 (m, 42H); 0.86 (t, 9H).

I. Compound 8: Heptadecan-9-yl8-((3-(1H-imidazol-1-yl)propyl)(nonyl)amino)octanoate Step 1:Heptadecan-9-yl 8-((3-chloropropyl)(nonyl)amino)octanoate

To a 0° C. solution of heptadecan-9-yl8-((3-hydroxypropyl)(nonyl)amino)octanoate (0.53 g, 0.91 mmol) in 4 mLof DCM was added mesyl chloride (0.070 mL, 0.91 mmol) followed bytriethylamine (0.13 mL, 0.91 mmol). The reaction was allowed to slowlywarm to rt and stir overnight. The reaction was quenched by the additionof water (˜10 mL). The mixture was extracted with DCM three times andthe pooled organics were washed with brine, dried over MgSO₄, filteredand concentrated in vacuo. The crude oil was purified by silica gelchromatography to afford heptadecan-9-yl8-((3-chloropropyl)(nonyl)amino)octanoate (0.23 g, 42%). ¹H NMR (300MHz, CDCl₃) δ: ppm 4.84 (p, 1H); 3.58 (t, 2H); 2.51 (br. s, 2H); 2.35(br. s, 2H); 2.26 (2, 2H); 1.86 (br. s, 2H); 1.40-1.60 (br. m, 12H);1.24 (br. m, 42H); 0.86 (t, 9H).

Step 2: Heptadecan-9-yl8-((3-(1H-imidazol-1-yl)propyl)(nonyl)amino)octanoate

In a round bottom flask, heptadecan-9-yl8-((3-chloropropyl)(nonyl)amino)octanoate (50 mg, 0.083 mmol) wascombined with imidazole (17 mg, 0.25 mmol), K₂CO₃ (35 mg, 0.25 mmol) inMeCN (0.5 mL). The flask was fitted with a condenser and placed in an82° C. heating mantle and was allowed to stir for 24 h. After this time,the reaction was allowed to cool to rt, was filtered and the filtratewas concentrated in vacuo. The crude oil was purified by silica gelchromatography (0-100% [DCM, 20% MeOH, 1% NH₄OH]/MeOH) to afford thedesired product as a clear oil (39 mg, 74%). UPLC/ELSD: RT=2.92 min. MS(ES): m/z (MH⁺) 633.994 for C₄₀H₇₇N₃O₂. ¹H NMR (300 MHz, CDCl₃) δ: ppm7.46 (s, 1H); 7.05 (s, 1H); 6.91 (s, 1H); 4.84 (dt, 1H); 4.02 (br. s,2H); 2.47 (br. s, 4H); 2.26 (t, 2H); 2.00 (br. s, 2H); 1.47-1.59 (br. m,10H); 1.24 (br. m, 44H); 0.86 (t, 9H).

J. Compound 9: Heptadecan-9-yl8-((2-acetoxyethyl)(8-(nonyloxy)-8-oxooctyl)amino) octanoate

To a solution of heptadecan-9-yl8-((2-hydroxyethyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate (100 mg,0.14 mmol) and acetic acid (8 mg, 0.13 mmol) in dichloromethane (1 mL)were added N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride(31 mg, 0.16 mmol), N,N-diisopropylethylamine (73 mg, 0.56 mmol) andDMAP (3 mg, 0.02 mmol). The reaction was allowed to stir at rt for 18 h.The reaction was diluted with dichloromethane and washed with saturatedsodium bicarbonate. The organic layer was separated and washed withbrine and dried over MgSO₄. The organic layer was filtered andevaporated in vacuo. The residue was purified by silica gelchromatography (0-100% (mixture of 1% NH₄OH, 20% MeOH indichloromethane) in dichloromethane) to yield heptadecan-9-yl8-((2-acetoxyethyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate (63 mg, 0.08mmol). UPLC/ELSD: RT=3.63 min. MS (ES): m/z (MH⁺) 753.07 for C₄₆H₈₉NO₆.¹H NMR (300 MHz, CDCl₃) δ: ppm 4.87 (p, 1H); 4.17-3.99 (m, 4H); 2.67 (m,2H); 2.43 (m, 3H); 2.29 (m, 4H); 2.05 (s, 3H); 1.71-1.17 (br. m, 63H);0.88 (m, 9H).

K. Compound 10: Heptadecan-9-yl8-((2-hydroxypropyl)(8-(nonyloxy)-8-oxooctyl)amino) octanoate

Compound 10 was synthesized according to the general procedure andRepresentative Procedure 1 described above. UPLC/ELSD: RT=3.73 min. MS(ES): m/z (MH⁺) 725.10 for C₄₅H₈₉NO₅. ¹H NMR (300 MHz, CDCl₃) δ: ppm4.86 (p, 1H); 4.05 (t, 2H); 3.80-3.54 (br. m, 1H); 2.61-2.13 (br. m,9H); 1.69-1.03 (br. m, 67H); 0.88 (m, 9H).

L. Compound 11: Heptadecan-9-yl(R)-8-((2-hydroxypropyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate

Compound 11 was synthesized according to the general procedure andRepresentative Procedure 1 described above. UPLC/ELSD: RT=5.21 min. MS(ES): m/z (MH⁺) 725.02 for C₄₅H₈₉NO₅. ¹H NMR (300 MHz, CDCl₃) δ: ppm4.86 (p, 1H); 4.05 (t, 2H); 3.72 (br. m, 1H); 2.65-2.10 (br. m, 8H);1.71-0.99 (br. m, 68H); 0.88 (m, 9H).

M. Compound 12: Heptadecan-9-yl(S)-8-((2-hydroxypropyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate

Compound 12 was synthesized according to the general procedure andRepresentative Procedure 1 described above. UPLC/ELSD: RT=5.30 min. MS(ES): m/z (MH⁺) 725.10 for C₄₅H₈₉NO₅. ¹H NMR (300 MHz, CDCl₃) δ: ppm4.86 (p, 1H); 4.05 (t, 2H); 3.71 (br. m, 1H); 2.64-2.10 (br. m, 8H);1.71-1.03 (br. m, 68H); 0.88 (m, 9H).

N. Compound 13: Heptadecan-9-yl8-((2-hydroxybutyl)(8-(nonyloxy)-8-oxooctyl)amino) octanoate

Compound 13 was synthesized according to the general procedure andRepresentative Procedure 1 described above. UPLC/ELSD: RT=3.89 min. MS(ES): m/z (MH⁺) 739.21 for C₄₆H₉₁NO₅. ¹H NMR (300 MHz, CDCl₃) δ: ppm4.86 (p, 1H); 4.05 (t, 2H); 3.58-3.38 (br. m, 1H); 2.65-2.15 (br. m,9H); 1.72-1.12 (br. m, 66H); 0.98 (t, 3H); 0.88 (m, 9H).

O. Compound 14: Heptadecan-9-yl8-((2-(dimethylamino)ethyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate

Compound 14 was synthesized according to the general procedure andRepresentative Procedure 1 described above. UPLC/ELSD: RT=3.51 min. MS(ES): m/z (MH⁺) 738.23 for C₄₆H₉₂N₂O₄. ¹H NMR (300 MHz, CDCl₃) δ: ppm4.84 (p, 1H); 4.04 (t, 2H); 2.95 (m, 2H); 2.78 (m, 6H); 2.44 (s, 6H);2.28 (m, 4H); 1.70-1.41 (br. m, 14H); 1.41-1.14 (br. m, 48H); 0.87 (m,9H).

P. Compound 15: Heptadecan-9-yl8-((2-methoxyethyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate

Compound 15 was synthesized according to the general procedure andRepresentative Procedure 1 described above. UPLC/ELSD: RT=3.90 min. MS(ES): m/z (MH⁺) 725.19 for C₄₅H₈₉NO₅. ¹H NMR (300 MHz, CDCl₃) δ: ppm4.86 (p, 1H); 4.05 (t, 2H); 3.43 (m, 2H); 3.34 (s, 3H); 2.61 (m, 2H);2.43 (m, 3H); 2.29 (m, 4H); 1.70-1.15 (br. m, 63H); 0.88 (m, 9H).

Q. Compound 16: Heptadecan-9-yl8-((3-methoxypropyl)(8-(nonyloxy)-8-oxooctyl)amino) octanoate

Compound 16 was synthesized according to the general procedure andRepresentative Procedure 1 described above. UPLC/ELSD: RT=3.90 min. MS(ES): m/z (MH⁺) 739.13 for C₄₆H₉₁NO₅. ¹H NMR (300 MHz, CDCl₃) δ: ppm4.89 (p, 1H); 4.08 (t, 2H); 3.42 (m, 2H); 3.35 (s, 3H); 2.55-2.21 (m,9H); 1.81-1.18 (br. m, 65H); 0.88 (m, 9H).

R. Compound 17: Heptadecan-9-yl 8-((2-(2-(dimethylamino)ethoxy)ethyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate

Compound 17 was synthesized according to the general procedure andRepresentative Procedure 1 described above. UPLC/ELSD: RT=3.72 min. MS(ES): m/z (MH⁺) 782.27 for C₄₈H₉₆N₂O₅. ¹H NMR (300 MHz, CDCl₃) δ: ppm4.88 (p, 1H); 4.08 (t, 2H); 3.57 (m, 4H); 2.72 (m, 2H); 2.52 (m, 5H);2.38-2.13 (br. m, 12H); 1.73-1.19 (br. m, 61H); 0.90 (m, 9H).

S. Compound 18: Heptadecan-9-yl8-((2-hydroxyethyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate

Compound 18 was synthesized according to the general procedure andRepresentative Procedure 1 described above or according to the schemebelow:

UPLC/ELSD: RT=3.59 min. MS (ES): m/z (MH⁺) 710.89 for C₄₄H₈₇NO₅. ¹H NMR(300 MHz, CDCl₃) δ: ppm 4.86 (m, 1H); 4.05 (t, 2H); 3.53 (br. m, 2H);2.83-2.36 (br. m, 5H); 2.29 (m, 4H); 0.96-1.71 (m, 64H); 0.88 (m, 9H).

T. Compound 19: Heptadecan-9-yl8-((3-hydroxypropyl)(8-(nonyloxy)-8-oxooctyl)amino) octanoate

Compound 19 was synthesized according to the general procedure andRepresentative Procedure 1 described above. UPLC/ELSD: RT=4.51 min. MS(ES): m/z (MH⁺) 725.19 for C₄₅H₈₉NO₅. ¹H NMR (300 MHz, CDCl₃) δ: ppm4.86 (p, 1H); 4.05 (t, 2H); 3.80 (m, 2H); 2.92-2.36 (br. m, 5H); 2.29(m, 4H); 1.89-1.42 (br. m, 16H); 1.42-1.02 (br. m, 50H); 0.88 (m, 9H).

U. Compound 20: Heptadecan-9-yl8-((4-hydroxybutyl)(8-(nonyloxy)-8-oxooctyl)amino) octanoate

Compound 20 was synthesized according to the general procedure andRepresentative Procedure 1 described above. UPLC/ELSD: RT=3.84 min. MS(ES): m/z (MH⁺) 739.21 for C₄₆H₉₁NO₅. ¹H NMR (300 MHz, CDCl₃) δ: ppm4.86 (p, 1H); 4.05 (t, 2H); 3.77-3.45 (br. m, 2H); 2.63-2.20 (br. m,8H); 1.82-1.40 (br. m, 18H); 1.40-1.15 (br. m, 51H); 0.88 (m, 9H).

V. Compound 21: Heptadecan-9-yl8-((2-cyanoethyl)(8-(nonyloxy)-8-oxooctyl)amino) octanoate

Compound 21 was synthesized according to the general procedure andRepresentative Procedure 1 described above. UPLC/ELSD: RT=4.04 min. MS(ES): m/z (MH⁺) 720.18 for C₄₅H₈₆N₂O₄. ¹H NMR (300 MHz, CDCl₃) δ: ppm4.88 (p, 1H); 4.07 (t, 2H); 2.81 (m, 2H); 2.44 (m, 5H); 2.30 (m, 4H);1.73-1.18 (br. m, 63H); 0.89 (m, 9H).

W. Compound 22: Heptadecan-9-yl8-((2-hydroxycyclohexyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate

Compound 22 was synthesized according to the general procedure andRepresentative Procedure 1 described above. UPLC/ELSD: RT=4.54 min. MS(ES): m/z (MH⁺) 765.21 for C₄₈H₉₃NO₅. ¹H NMR (300 MHz, CDCl₃) δ: ppm4.86 (p, 1H); 4.05 (t, 2H); 2.89-2.34 (br. m, 4H); 2.28 (m, 4H); 2.00(m, 1H); 1.86-0.99 (br. m, 72H); 0.88 (m, 9H).

X. Compound 23: Heptadecan-9-yl10-((2-hydroxyethyl)(8-(nonyloxy)-8-oxooctyl)amino) decanoate

Compound 23 was synthesized according to the general procedure andRepresentative Procedure 1 described above. UPLC/ELSD: RT=3.75 min. MS(ES): m/z (MH⁺) 739.13 for C₄₆H₉₁NO₅. ¹H NMR (300 MHz, CDCl₃) δ: ppm4.86 (m, 1H); 4.05 (m, 2H); 3.72-3.46 (br. m, 2H); 2.81-2.35 (br. m,5H); 2.29 (m, 4H); 1.71-1.40 (br. m, 13H); 1.40-1.15 (br. m, 55H); 0.88(m, 9H).

Y. Compound 24: Heptadecan-9-yl (Z)-8_((2-hydroxyethyl)(8-(non-2-en-1-yloxy)-8-oxooctyl)amino)octanoate

Compound 24 was synthesized according to the general procedure andRepresentative Procedure 1 described above. UPLC/ELSD: RT=3.54 min. MS(ES): m/z (MH⁺) 708.95 for C₄₄H₈₅NO₅. ¹H NMR (300 MHz, CDCl₃) δ: ppm5.74-5.44 (br. m, 2H); 4.86 (m, 1H); 4.62 (m, 2H); 3.71-3.40 (br. m,2H); 2.81-2.37 (br. m, 5H); 2.29 (m, 4H); 2.09 (m, 2H); 1.70-1.14 (br.m, 58H); 0.88 (m, 9H).

Z. Compound 25: Heptadecan-9-yl8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate

Compound 25 was synthesized according to the general procedure andRepresentative Procedure 1 described above. UPLC/ELSD: RT=3.66 min. MS(ES): m/z (MH⁺) 711.00 for C₄₄H₈₇NO₅. ¹H NMR (300 MHz, CDCl₃) δ: ppm4.86 (m, 1H); 4.05 (t, 2H); 3.68-3.46 (br. m, 2H); 2.77-2.37 (br. m,5H); 2.29 (m, 4H); 1.74-1.41 (br. m, 14H); 1.39-1.18 (m, 50H); 0.88 (m,9H).

AA. Compound 26: Heptadecan-9-yl8-((2-hydroxyethyl)(4-(nonyloxy)-4-oxobutyl)amino) octanoate

Compound 26 was synthesized according to the general procedure andRepresentative Procedure 1 described above. UPLC/ELSD: RT=4.29 min. MS(ES): m/z (MH⁺) 655.07 for C₄₀H₇₉NO₅. ¹H NMR (300 MHz, CDCl₃) δ: ppm4.86 (p, 1H); 4.06 (t, 2H); 3.79 (br. m, 2H); 2.91-2.20 (br. m, 10H);1.98-1.03 (br. m, 55H); 0.88 (m, 9H).

AB. Compound 27: Nonyl8-((6-(heptadecan-9-yloxy)-6-oxohexyl)(2-hydroxyethyl)amino) octanoate

Compound 27 was synthesized according to the general procedure andRepresentative Procedure 1 described above. UPLC/ELSD: RT=3.57 min. MS(ES): m/z (MH⁺) 683.12 for C₄₂H₈₃NO₅. ¹H NMR (300 MHz, CDCl₃) δ: ppm4.86 (m, 1H); 4.05 (m, 2H); 3.70-3.45 (br. m, 2H); 2.78-2.35 (br. m,5H); 2.29 (m, 4H); 1.73-1.41 (m, 13H); 1.41-1.16 (m, 47H); 0.88 (m, 9H).

AC. Compound 28: Heptadecan-9-yl8-((8-((2-hexylcyclopropyl)methoxy)-8-oxooctyl)(2-hydroxyethyl)amino)octanoate

Compound 28 was synthesized according to the general procedure andRepresentative Procedure 1 described above using Intermediate C.UPLC/ELSD: RT=5.17 min. MS (ES): m/z (MH⁺) 722.97 for C₄₅H₈₇NO₅. ¹H NMR(300 MHz, CDCl₃) δ: ppm 4.86 (p, 1H); 4.17 (m, 1H); 3.93 (m, 1H); 3.61(br. m, 2H); 2.97-2.37 (br. m, 6H); 2.35-2.21 (m, 4H); 1.74-0.97 (br. m,60H); 0.94-0.79 (m, 10H); 0.74 (m, 1H); 0.01 (m, 1H).

AD. Compound 29: Di(heptadecan-9-yl)8,8′-((2-hydroxyethyl)azanediyl)dioctanoate

Compound 29 was synthesized according to the general procedure andRepresentative Procedure 1 described above. UPLC/ELSD: RT=3.98 min. MS(ES): m/z (MH⁺) 823.19 for C₅₂H₁₀₃NO₅. ¹H NMR (300 MHz, CDCl₃) δ: ppm4.86 (m, 2H); 3.72-3.44 (br. m, 2H); 2.83-2.34 (br. m, 5H); 2.28 (m,4H); 1.69-1.39 (br. m, 16H); 1.39-1.16 (br. m, 62H); 0.88 (m, 12H).

AE. Compound 30:7-((2-Hydroxyethyl)(8-(nonyloxy)-8-oxooctyl)amino)heptyl2-octyldecanoate

Compound 30 was synthesized according to the general procedure andRepresentative Procedure 1 described above using Intermediate B.UPLC/ELSD: RT=3.55 min. MS (ES): m/z (MH⁺) 711.16 for C₄₄H₈₇NO₅. ¹H NMR(300 MHz, CDCl₃) δ: ppm 4.06 (m, 4H); 3.69-3.44 (br. m, 2H); 2.71-2.39(br. m, 5H); 2.29 (m, 3H); 1.70-1.16 (br. m, 64H); 0.88 (m, 9H).

AF. Compound 31: heptadecan-9-yl(Z)-8-((2-hydroxyethyl)(octadec-9-en-1-yl)amino) octanoate

Compound 31 was synthesized according to the general procedure andRepresentative Procedure 1 described above. UPLC/ELSD: RT=3.83 min. MS(ES): m/z (MH⁺) 693.20 for C₄₅H₈₉NO₃. ¹H NMR (300 MHz, CDCl₃) δ: ppm5.37 (m, 2H); 4.89 (p, 1H); 3.58 (br. m, 2H); 2.72-2.43 (br. m, 5H);2.30 (m, 2H), 2.05 (m, 4H); 1.71-1.03 (br. m, 63H), 0.90 (m, 9H).

AG. Compound 32: nonyl8-((2-hydroxyethyl)(8-oxo-8-(pentadecan-7-yloxy)octyl)amino) octanoate

Compound 32 was synthesized according to the general procedure andRepresentative Procedure 1 described above. UPLC/ELSD: RT=3.45 min. MS(ES): m/z (MH⁺) 683.20 for C₄₂H₈₃NO₅. ¹H NMR (300 MHz, CDCl₃) δ: ppm4.89 (p, 1H); 4.08 (t, 2H); 3.60 (br. m, 2H); 2.85-2.40 (br. m, 5H);2.31 (m, 4H), 1.78-1.01 (m, 59H), 0.90 (m, 9H).

AH. Compound 33: nonyl8-((2-hydroxyethyl)(8-oxo-8-(tetradecan-6-yloxy)octyl)amino) octanoate

Compound 33 was synthesized according to the general procedure andRepresentative Procedure 1 described above. UPLC/ELSD: RT=3.39 min. MS(ES): m/z (MH⁺) 669.09 for C₄₁H₈₁NO₅. ¹H NMR (300 MHz, CDCl₃) δ: ppm4.89 (p, 1H); 4.08 (t, 2H); 3.84-3.54 (br. m, 2H); 2.99-2.41 (br. m,5H); 2.31 (m, 4H), 1.76-1.02 (br. m, 57H), 0.90 (m, 9H).

AI. Compound 34: dodecan-4-yl8-((2-hydroxyethyl)(8-(nonyloxy)-8-oxooctyl)amino) octanoate

Compound 34 was synthesized according to the general procedure andRepresentative Procedure 1 described above. UPLC/ELSD: RT=3.21 min. MS(ES): m/z (MH⁺) 641.05 for C₃₉H₇₇NO₅. ¹H NMR (300 MHz, CDCl₃) δ: ppm4.91 (p, 1H); 4.08 (t, 2H); 3.67 (br. m, 2H); 3.03-2.44 (br. m, 5H);2.30 (m, 4H), 1.75-1.00 (br. m, 53H), 0.90 (m, 9H).

AJ. Compound 35: nonyl8-((2-hydroxyethyl)(8-oxo-8-(undecan-3-yloxy)octyl)amino) octanoate

Compound 35 was synthesized according to the general procedure andRepresentative Procedure 1 described above. UPLC/ELSD: RT=3.16 min. MS(ES): m/z (MH⁺) 627.11 for C₃₈H₇₅NO₅. ¹H NMR (300 MHz, CDCl₃) δ: ppm4.83 (p, 1H); 4.08 (t, 2H); 3.63 (br. m, 2H); 2.81-2.39 (br. m, 5H);2.31 (m, 4H), 1.74-1.01 (br. m, 51H), 0.90 (m, 9H).

AK. Compound 36: decan-2-yl8-((2-hydroxyethyl)(8-(nonyloxy)-8-oxooctyl)amino) octanoate

Compound 36 was synthesized according to the general procedure andRepresentative Procedure 1 described above. UPLC/ELSD: RT=3.05 min. MS(ES): m/z (MH⁺) 613.00 for C₃₇H₇₃NO₅. ¹H NMR (300 MHz, CDCl₃) δ: ppm4.91 (p, 1H); 4.08 (t, 2H); 3.55 (m, 2H); 2.60 (m, 2H); 2.47 (m, 4H);2.29 (m, 4H), 1.731-1.01 (m, 51H), 0.90 (m, 6H).

AL. Compound 47: heptadecan-9-yl8-((2-hydroxyethyl)(8-(2-octylcyclopropyl)octyl)amino)octanoate

Compound 47 was synthesized according to the general procedure andRepresentative Procedure 1 described above. UPLC/ELSD: RT=3.92 min. MS(ES): m/z (MH⁺) 707.39 for C₄₆H₉₁NO₃. ¹H NMR (300 MHz, CDCl₃) δ: ppm4.86 (p, 1H); 3.56 (br. m, 2H); 2.72-2.38 (br. m, 5H); 2.28 (t, 2H);1.70-1.02 (br. m, 67H), 0.88 (m, 9H); 0.71-0.49 (m, 4H); −0.33 (m, 1H).

AM. Compound 48: decan-2-yl8-((8-(heptadecan-9-yloxy)-8-oxooctyl)(2-hydroxyethyl)amino)octanoate

Compound 48 was synthesized according to the general procedure andRepresentative Procedure 1 described above. UPLC/ELSD: RT=3.60 min. MS(ES): m/z (MH⁺) 725.10 for C₄₅H₈₉NO₅. ¹H NMR (300 MHz, CDCl₃) δ: ppm4.91 (m, 2H); 3.59 (br. m, 2H); 2.79-2.37 (br. m, 5H); 2.29 (m, 4H);1.74-1.13 (m, 66H); 0.90 (m, 9H).

AN. Compound 49: heptadecan-9-yl8-((2-hydroxyethyl)(8-oxo-8-(undecan-3-yloxy)octyl)amino)octanoate

Compound 49 was synthesized according to the general procedure andRepresentative Procedure 1 described above. UPLC/ELSD: RT=3.68 min. MS(ES): m/z (MH⁺) 739.21 for C₄₆H₉₁NO₅. ¹H NMR (300 MHz, CDCl₃) δ: ppm4.89 (m, 2H); 3.56 (br. m, 2H); 2.68-2.39 (br. m, 5H); 2.30 (m, 4H);1.71-1.19 (m, 66H); 0.90 (m, 12H).

AO. Compound 50: dodecan-4-yl8-((8-(heptadecan-9-yloxy)-8-oxooctyl)(2-hydroxyethyl)amino)octanoate

Compound 50 was synthesized according to the general procedure andRepresentative Procedure 1 described above. UPLC/ELSD: RT=3.73 min. MS(ES): m/z (MH⁺) 753.23 for C₄₇H₉₃NO₅. ¹H NMR (300 MHz, CDCl₃) δ: ppm4.89 (m, 2H); 3.60 (br. m, 2H); 2.75-2.43 (br. m, 5H); 2.30 (m, 4H);1.71-1.44 (m, 16H); 1.28 (m, 51H); 0.90 (m, 12H).

AP. Compound 51: heptadecan-9-yl8-((4-butoxy-4-oxobutyl)(2-hydroxyethyl)amino) octanoate

Compound 51 was synthesized according to the general procedure andRepresentative Procedure 1 described above. UPLC/ELSD: RT=3.05 min. MS(ES): m/z (MH⁺) 584.87 for C₃₅H₆₉NO₅. ¹H NMR (300 MHz, CDCl₃) δ: ppm4.89 (p, 1H); 4.10 (t, 2H); 3.61 (br. m, 2H); 2.81-2.21 (br. m, 9H);1.87 (br. m, 2H), 1.70-1.04 (m, 43H), 0.98-0.82 (m, 9H).

AQ. Compound 52: heptadecan-9-yl8-((2-hydroxyethyl)(4-oxo-4-(pentyloxy)butyl)amino) octanoate

Compound 52 was synthesized according to the general procedure andRepresentative Procedure 1 described above. UPLC/ELSD: RT=3.11 min. MS(ES): m/z (MH⁺) 598.90 for C₃₆H₇₁NO₅. ¹H NMR (300 MHz, CDCl₃) δ: ppm4.89 (p, 1H); 4.09 (t, 2H); 3.61 (br. m, 2H); 2.89-2.22 (br. m, 9H);1.87 (br. m, 2H), 1.73-1.43 (m, 11H), 1.28 (m, 34H); 0.90 (m, 9H).

AR. Compound 53: heptadecan-9-yl8-((4-(hexyloxy)-4-oxobutyl)(2-hydroxyethyl)amino) octanoate

Compound 53 was synthesized according to the general procedure andRepresentative Procedure 1 described above. UPLC/ELSD: RT=3.22 min. MS(ES): m/z (MH⁺) 612.92 for C₃₇H₇₃NO₅. ¹H NMR (300 MHz, CDCl₃) δ: ppm4.86 (p, 1H); 4.06 (t, 2H); 3.55 (br. m, 2H); 2.68-2.38 (br. m, 5H);2.28 (m, 4H); 1.79 (br. m, 2H); 1.71-0.96 (m, 48H); 0.88 (m, 9H).

AS. Compound 54: heptadecan-9-yl8-((4-(heptyloxy)-4-oxobutyl)(2-hydroxyethyl)amino) octanoate

Compound 54 was synthesized according to the general procedure andRepresentative Procedure 1 described above. UPLC/ELSD: RT=3.28 min. MS(ES): m/z (MH⁺) 626.94 for C₃₈H₇₅NO₅. ¹H NMR (300 MHz, CDCl₃) δ: ppm4.89 (p, 1H); 4.09 (t, 2H); 3.60 (br. m, 2H); 2.77-2.42 (br. m, 5H);2.32 (m, 4H); 1.84 (br. m, 2H); 1.75-1.03 (m, 49H); 0.90 (m, 9H).

AT. Compound 55: heptadecan-9-yl8-((4-((2-hexylcyclopropyl)methoxy)-4-oxobutyl)(2-hydroxyethyl)amino)octanoate

Compound 55 was synthesized according to the general procedure andRepresentative Procedure 1 described above. UPLC/ELSD: RT=3.37 min. MS(ES): m/z (MH⁺) 667.04 for C₄₁H₇₉NO₅. ¹H NMR (300 MHz, CDCl₃) δ: ppm4.83 (p, 1H); 4.15 (m, 1H); 3.95 (m, 1H); 3.53 (br. m, 2H); 2.66-2.39(br. m, 5H); 2.34-2.19 (m, 4H); 1.78 (br. m, 2H); 1.66-0.98 (m, 50H);0.85 (m, 10H); 0.70 (m, 1H); 0.00 (m, 1H).

AU. Compound 56: nonyl8-((2-hydroxyethyl)(8-oxo-8-(tridecan-7-yloxy)octyl)amino) octanoate

Compound 56 was synthesized according to the general procedure andRepresentative Procedure 1 described above. UPLC/ELSD: RT=3.28 min. MS(ES): m/z (MH⁺) 654.99 for C₄₀H₇₉NO₅. ¹H NMR (300 MHz, CDCl₃) δ: ppm4.89 (p, 1H); 4.08 (t, 2H); 3.60 (br. m, 2H); 2.77-2.40 (br. m, 5H);2.30 (m, 4H); 1.78-0.99 (m, 55H); 0.90 (m, 9H).

AV. Compound 57: nonan-5-yl8-((2-hydroxyethyl)(8-(nonyloxy)-8-oxooctyl)amino) octanoate

Compound 57 was synthesized according to the general procedure andRepresentative Procedure 1 described above. UPLC/ELSD: RT=2.88 min. MS(ES): m/z (MH⁺) 598.98 for C₃₆H₇₁NO₅. ¹H NMR (300 MHz, CDCl₃) δ: ppm4.89 (p, 1H); 4.08 (t, 2H); 3.59 (br. m, 2H); 2.82-2.37 (br. m, 5H);2.31 (m, 4H); 1.73-1.03 (m, 47H); 0.91 (m, 9H).

AW. Compound 58: heptan-4-yl8-((2-hydroxyethyl)(8-(nonyloxy)-8-oxooctyl)amino) octanoate

Compound 58 was synthesized according to the general procedure andRepresentative Procedure 1 described above. UPLC/ELSD: RT=2.67 min. MS(ES): m/z (MH⁺) 570.93 for C₃₄H₆₇NO₅. ¹H NMR (300 MHz, CDCl₃) δ: ppm4.93 (p, 1H); 4.08 (t, 2H); 3.57 (br. m, 2H); 2.69-2.42 (br. m, 5H);2.30 (m, 4H); 1.72-1.04 (m, 43H); 0.93 (m, 9H).

AX. Compound 59: nonyl8-((2-hydroxyethyl)(8-oxo-8-(pentan-3-yloxy)octyl)amino) octanoate

Compound 59 was synthesized according to the general procedure andRepresentative Procedure 1 described above. UPLC/ELSD: RT=2.39 min. MS(ES): m/z (MH⁺) 542.80 for C₃₂H₆₃NO₅. ¹H NMR (300 MHz, CDCl₃) δ: ppm4.78 (p, 1H); 4.08 (t, 2H); 3.57 (br. m, 2H); 2.71-2.39 (br. m, 5H);2.31 (m, 4H); 1.77-1.05 (m, 39H); 0.90 (m, 9H).

AY. Compound 60: (5Z,12Z)-Heptadeca-5,12-dien-9-yl8-((2-hydroxyethyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate(5Z,12Z)-Heptadeca-5,12-dien-9-ol

To a solution of (Z)-1-bromooct-3-ene (6.2 g, 32.4 mmol) in THF (45 mL)Mg turnings were added (0.843 g, 34.7 mmol). The reaction was heated to45° C. for 3 h. The reaction was cooled to 0° C. and ethyl formate (2.4g, 32.4 mmol) in THF (5 mL) was added dropwise. The reaction was allowedto warm to rt and stir for 30 min. The reaction was cooled to 0° C. andquenched with water (15 mL) and 6N HCl (15 mL). The reaction was stirreduntil all the Mg was dissolved. Water (25 mL) was added and the mixturewas extracted with hexanes (3×25 mL). The combined organic layer waswashed with brine, separated, dried over Na₂SO₄, filtered, andevaporated under vacuum. The residue was dissolved in EtOH (20 mL), asolution of KOH in water (1.76 g in 8 mL of water) was added and allowedto stir for 15 min. EtOH was evaporated under vacuum. The residue wasdiluted with water (20 mL), acidified with 6N HCl (20 mL) and extractedwith hexanes (3×). The combined organic layers were washed with brine,separated, dried over Na₂SO₄, filtered, and evaporated under vacuum. Theresidue was purified by silica gel chromatography with (0-5%) EtOAc inhexanes to obtain (5Z,12Z)-heptadeca-5,12-dien-9-ol (2.3 g, 9.1 mmol,28%). ¹H NMR (300 MHz, CDCl₃) δ: ppm 5.41 (m, 4); 3.66 (m, 1H); 2.13 (m,8H); 1.51 (m, 5H); 1.36 (m, 8H); 0.92 (m, 6H).

(5Z,12Z)-Heptadeca-5,12-dien-9-yl8-((2-hydroxyethyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate

Compound 60 was synthesized according to the general procedure andRepresentative Procedure 1 described above. UPLC/ELSD: RT=3.36 min. MS(ES): m/z (MH⁺) 707.10 for C₄₄H₈₃NO₅. ¹H NMR (300 MHz, CDCl₃) δ: ppm5.37 (m, 4H); 4.92 (p, 1H); 4.08 (t, 2H); 3.57 (br. m, 2H); 2.73-2.38(br. m, 5H); 2.31 (m, 4H); 2.04 (m, 8H); 1.73-1.01 (m, 47H); 0.92 (m,9H).

AZ. Compound 61: (5Z,12Z)-heptadeca-5,12-dien-9-yl8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate

Compound 61 was synthesized according to the general procedure andRepresentative Procedure 1 described above. UPLC/ELSD: RT=3.39 min. MS(ES): m/z (MH⁺) 707.10 for C₄₄H₈₃NO₅. ¹H NMR (300 MHz, CDCl₃) δ: ppm5.37 (m, 4H); 4.92 (p, 1H); 4.08 (t, 2H); 3.58 (br. m, 2H); 2.70-2.41(br. m, 5H); 2.32 (m, 4H); 2.04 (m, 8H); 1.77-1.03 (m, 47H); 0.92 (m,9H).

X1. Compound 65: 1-Cyclopropylnonyl8-((8-(heptadecan-9-yloxy)-8-oxooctyl)(2-hydroxyethyl)amino)octanoate

Compound 65 was synthesized according to the general procedure andRepresentative Procedure 1 described above. UPLC/ELSD: RT=3.72 min. MS(ES): m/z (MH⁺) 750.9 for C₄₇H₉₁NO₅. ¹H NMR (300 MHz, CDCl₃) δ: ppm 4.89(m, 1H); 4.28 (m, 1H); 3.54 (m, 2H); 2.59 (m, 2H); 2.46 (m, 4H); 2.29(m, 4H), 1.73-1.18 (m, 61H); 0.90 (m, 10H); 0.62-0.33 (m, 3H); 0.28 (m,1H).

X2. Compound 66: Heptadecan-9-yl8-((2-hydroxyethyl)(8-oxo-8-((4-pentylcyclohexyl)oxy)octyl)amino)octanoate

Compound 66 was synthesized according to the general procedure andRepresentative Procedure 1 described above. UPLC/ELSD: RT=3.72 min. MS(ES): m/z (MH⁺) 736.9 for C₄₆H₈₉NO₅. ¹H NMR (300 MHz, CDCl₃) δ: ppm 5.00(m, 0.5H); 4.89 (m, 1H); 4.68 (m, 0.6H); 3.56 (m, 2H), 2.61 (br. m, 2H);2.48 (m, 4H); 2.30 (m, 4H); 1.98 (m, 1H); 1.82 (m, 2H); 1.73-1.14 (m,61H); 1.04 (m, 1H); 0.90 (m, 9H).

X3. Compound 67: Heptadecan-9-yl8-((2-hydroxyethyl)(4-oxo-4-((4-pentylcyclohexyl)oxy)butyl)amino)octanoate

Compound 67 was synthesized according to the general procedure andRepresentative Procedure 1 described above. UPLC/ELSD: RT=3.56 min. MS(ES): m/z (MH⁺) 680.8 for C₄₂H₈₁NO₅. ¹H NMR (300 MHz, CDCl₃) δ: ppm 5.01(m, 0.4H); 4.89 (m, 1H); 4.68 (m, 0.6H); 3.59 (m, 2H), 2.72-2.43 (br. m,6H); 2.30 (m, 4H); 1.98 (m, 1H); 1.83 (m, 4H); 1.69-1.44 (m, 10H); 1.28(m, 41H); 1.03 (m, 1H); 0.90 (m, 9H).

X4. Compound 68: Heptadecan-9-yl8-((2-hydroxyethyl)(6-oxo-6-((4-pentylcyclohexyl)oxy)hexyl)amino)octanoate

Compound 68 was synthesized according to the general procedure andRepresentative Procedure 1 described above. UPLC/ELSD: RT=3.66 min. MS(ES): m/z (MH⁺) 708.9 for C₄₄H₈₅NO₅. ¹H NMR (300 MHz, CDCl₃) δ: ppm 5.00(m, 0.5H); 4.89 (m, 1H); 4.68 (m, 0.6H); 3.55 (m, 2H), 2.66-2.39 (br. m,6H); 2.30 (m, 4H); 1.97 (m, 1H); 1.83 (m, 2H); 1.73-1.41 (m, 15H);1.41-1.17 (m, 42H); 1.04 (m, 1H); 0.90 (m, 9H).

XX1. Compound 69: Heptadecan-9-yl8-((2,3-dihydroxypropyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate

Compound 69 was synthesized according to the general procedure andRepresentative Procedure 1 described above. UPLC/ELSD: RT=3.60 min. MS(ES): m/z (MH⁺) 741.0 for C₄₅H₈₉NO₆. ¹H NMR (300 MHz, CDCl₃) δ: ppm 4.89(p, 1H); 4.08 (t, 2H); 3.76 (br. m, 2H); 3.51 (m, 1H); 2.57 (m, 6H);2.31 (m, 4H); 1.71-1.41 (m, 14H); 1.41-1.12 (m, 48H); 0.90 (m, 9H).

XX2. Compound 70: Heptadecan-9-yl8-((4-(decan-2-yloxy)-4-oxobutyl)(2-hydroxyethyl)amino)octanoate

Compound 70 was synthesized according to the general procedure andRepresentative Procedure 1 described above. UPLC/ELSD: RT=3.44 min. MS(ES): m/z (MH⁺) 668.9 for C₄₁H₈₁NO₅. ¹H NMR (300 MHz, CDCl₃) δ: ppm 4.91(m, 2H); 3.57 (m, 2H); 2.71-2.40 (m, 5H); 2.30 (m, 4H), 1.80 (m, 2H);1.71-1.40 (m, 11H); 1.39-1.05 (m, 45H); 0.90 (m, 9H).

X5. Compound 71: Heptadecan-9-yl8-((2-hydroxyethyl)(4-oxo-4-(tetradecan-6-yloxy)butyl)amino)octanoate

Compound 71 was synthesized according to the general procedure andRepresentative Procedure 1 described above. UPLC/ELSD: RT=3.72 min. MS(ES): m/z (MH⁺) 724.9 for C₄₅H₈₉NO₅. ¹H NMR (300 MHz, CDCl₃) δ: ppm 4.89(m, 2H); 3.56 (m, 2H); 2.70-2.41 (m, 6H); 2.33 (m, 4H), 1.80 (m, 2H);1.69-1.41 (m, 13H); 1.28 (m, 48H); 0.90 (m, 12H).

XX3. Compound 72: Heptadecan-9-yl8-((2-hydroxyethyl)(4-oxo-4-(undecan-3-yloxy)butyl)amino)octanoate

Compound 72 was synthesized according to the general procedure andRepresentative Procedure 1 described above. UPLC/ELSD: RT=3.57 min. MS(ES): m/z (MH⁺) 683.0 for C₄₂H₈₃NO₅. ¹H NMR (300 MHz, CDCl₃) δ: ppm 4.86(m, 2H); 3.58 (br. m, 2H); 2.75-2.41 (br. m, 5H); 2.30 (m, 4H), 1.81(br. m, 2H); 1.70-1.42 (m, 13H); 1.40-1.18 (m, 42H); 0.90 (m, 12H).

X6. Compound 73: Heptadecan-9-yl8-((2-hydroxyethyl)(4-oxo-4-(pentadecan-7-yloxy)butyl)amino)octanoate

Compound 73 was synthesized according to the general procedure andRepresentative Procedure 1 described above. UPLC/ELSD: RT=3.80 min. MS(ES): m/z (MH⁺) 739.09 for C₄₆H₉₁NO₅. ¹H NMR (300 MHz, CDCl₃) δ: ppm4.89 (m, 2H); 3.59 (br. m, 2H); 2.81-2.43 (br. m, 6H); 2.31 (m, 4H);1.83 (m, 2H); 1.69-1.42 (m, 12H); 1.28 (m, 50H); 0.90 (m, 12H).

X7. Compound 74: Heptadecan-9-yl8-((4-(dodecan-4-yloxy)-4-oxobutyl)(2-hydroxyethyl)amino)octanoate

Compound 74 was synthesized according to the general procedure andRepresentative Procedure 1 described above. UPLC/ELSD: RT=3.68 min. MS(ES): m/z (MH⁺) 696.9 for C₄₃H₈₅NO₅. ¹H NMR (300 MHz, CDCl₃) δ: ppm 4.89(m, 2H); 3.56 (m, 2H); 2.70-2.41 (m, 6H); 2.30 (m, 4H), 1.80 (m, 2H);1.70-1.40 (m, 12H); 1.28 (m, 44H); 0.90 (m, 12H).

XX4. Compound 75: Heptadecan-9-yl8-((2-hydroxyethyl)(6-oxo-6-(undecan-3-yloxy)hexyl)amino)octanoate

Compound 75 was synthesized according to the general procedure andRepresentative Procedure 1 described above. UPLC/ELSD: RT=3.67 min. MS(ES): m/z (MH⁺) 711.1 for C₄₄H₈₇NO₅. ¹H NMR (300 MHz, CDCl₃) δ: ppm 4.86(m, 2H); 3.57 (m, 2H); 2.72-2.40 (br. m, 5H); 2.30 (m, 4H); 1.70-1.42(m, 16H); 1.28 (m, 45H); 0.90 (m, 12H).

XX5. Compound 79: Nonyl8-((2-hydroxyethyl)(8-oxo-8-((4-pentylcyclohexyl)oxy)octyl)amino)octanoate

Compound 79 was synthesized according to the general procedure andRepresentative Procedure 1 described above. UPLC/ELSD: RT=3.10 min. MS(ES): m/z (MH⁺) 624.8 for C₃₈H₇₃NO₅. ¹H NMR (300 MHz, CDCl₃) δ: ppm 5.00(br. m, 0.5H); 4.68 (m, 0.5H); 4.08 (t, 2H); 3.56 (m, 2H); 2.72-2.38 (m,6H); 2.31 (m, 4H), 1.97 (m, 1H); 1.82 (m, 2H); 1.73-0.95 (m, 48H), 0.90(m, 6H).

XX6. Compound 80: [1,1′-Bi(cyclohexan)]-4-yl8-((2-hydroxyethyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate

Compound 80 was synthesized according to the general procedure andRepresentative Procedure 1 described above. UPLC/ELSD: RT=3.10 min. MS(ES): m/z (MH⁺) 636.9 for C₃₉H₇₃NO₅. ¹H NMR (300 MHz, CDCl₃) δ: ppm 5.01(br. m, 0.5H); 4.65 (m, 0.5H); 4.08 (t, 2H); 3.56 (m, 2H); 2.69-2.36 (m,6H); 2.31 (m, 4H); 2.07-0.84 (m, 57H).

XX7. Compound 81: Cyclopentadecyl8-((2-hydroxyethyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate

Compound 81 was synthesized according to the general procedure andRepresentative Procedure 1 described above. UPLC/ELSD: RT=3.36 min. MS(ES): m/z (MH⁺) 681.0 for C₄₂H₈₁NO₅. ¹H NMR (300 MHz, CDCl₃) δ: ppm 4.91(p, 1H); 4.08 (t, 2H); 3.57 (br. m, 2H); 2.74-2.39 (m, 6H); 2.30 (m,4H), 1.73-1.03 (m, 62H), 0.90 (m, 3H).

XX8. Compound 94: Heptadecan-9-yl)8-(benzyl(8-nonyloxy)-8-oxooctyl)amino)octanoate Heptadecan-9-yl8-(benylamino)octanoate

A solution of heptadecan-9-yl 8-bromooctanoate (250 mg, 0.542 mmol) inphenylmethanamine (1.2 mL, 10.83 mmol) was allowed to stir at rt for 6h. The reaction was cooled to rt and solvents were evaporated in vacuo.The residue was taken-up in ethyl acetate and washed with saturatedaqueous sodium bicarbonate. The organic layer was separated and washedwith brine, dried over Na₂SO₄ and evaporated in vacuo. The residue waspurified by silica gel chromatography (20-100% (mixture of 1% NH₄OH, 20%MeOH in dichloromethane) in dichloromethane) to obtain heptadecan-9-yl8-(benzylamino)octanoate (200 mg, 0.41 mmol, 76%). UPLC/ELSD: RT=2.87min. MS (ES): m/z (MH⁺) 488.4 for C₃₂H₅₇NO₂. ¹H NMR (300 MHz, CDCl₃) δ:ppm 7.35-7.25 (br. m, 5H); 4.89 (p, 1H); 3.81 (s, 2H); 2.65 (t, 2H);2.29 (t, 2H); 1.65-1.51 (br. m, 8H); 1.28 (m, 30H); 0.90 (m, 6H).

Heptadecan-9-yl 8-(benzyl(8-(nonyloxy)-8-oxooctyl)amino)octanoate

A solution of heptadecan-9-yl 8-(benylamino)octanoate (200 mg, 0.41mmol), nonyl 8-bromooctanoate (172 mg, 0.49 mmol) andN,N-diisopropylethylamine (100 μL, 0.57 mmol) were dissolved in ethanoland was allowed to stir at 62° C. for 48 h. The reaction was cooled tort and solvents were evaporated in vacuo. The residue was taken-up inethyl acetate and washed with saturated aqueous sodium bicarbonate. Theorganic layer was separated and washed with brine, dried over Na₂SO₄ andevaporated in vacuo. The residue was purified by silica gelchromatography (0-100% (mixture of 1% NH₄OH, 20% MeOH indichloromethane) in dichloromethane) to obtain heptadecan-9-yl8-(benzyl(8-(nonyloxy)-8-oxooctyl)amino)octanoate (138 mg, 0.18 mmol,45%). UPLC/ELSD: RT=3.78 min. MS (ES): m/z (MH⁺) 757.0 for C₄₉H₈₉NO₄. ¹HNMR (300 MHz, CDCl₃) δ: ppm 7.33-7.23 (br. m, 5H); 4.89 (p, 1H); 4.08(t, 2H); 3.55 (s, 2H); 2.40 (m, 4H); 2.30 (m, 4H); 1.64-1.28 (br. m,62H); 0.90 (m, 9H).

X8. Compound 96:7-((8-(Heptadecan-9-yloxy)-8-oxooctyl)(2-hydroxyethyl)amino)heptyldecanoate

Compound 96 was synthesized according to the general procedure andRepresentative Procedure 1 described above. UPLC/ELSD: RT=2.74 min. MS(ES): m/z (MH⁺) 711.0 for C₄₄H₈₇NO₅. ¹H NMR (300 MHz, CDCl₃) δ: ppm 4.89(m, 1H); 4.08 (t, 2H); 3.61 (m, 2H); 2.88-2.37 (br. m, 6H); 2.31 (m,4H), 1.79-1.04 (m, 62H); 0.90 (m, 9H).

X9. Compound 98:8-((8-(Heptadecan-9-yloxy)-8-oxooctyl)(2-hydroxyethyl)amino)octan-2-yldecanoate Octane-1,7-diol

A solution of 7-oxooctanoic acid (4 g, 25.29 mmol) in THF (10 mL) wasadded to a stirred solution of LAH in THF (70 mL) under N₂ at 0° C. Themixture was allowed to warm to rt and stir at rt for 4 h, after whichtime 10 mL of sat. Na₂SO₄.10H₂O (aq) was added to the solution slowly.White solid crashed out. Additional solid Na₂SO₄.10H₂O was added and themixture was filtered through a plug of celite. The filtrate was dilutedwith EtOAc and washed with brine. The organic layer was separated, driedover Na₂SO₄, filtered, and evaporated under vacuum. The residue waspurified by silica gel chromatography with (0-40%) EtOAc in hexanes toobtain octane-1,7-diol (2.97 g, 20.31 mmol, 80%). ¹H NMR (300 MHz,CDCl₃) δ: ppm 3.81 (m, 1H); 3.66 (t, 2H); 1.66-1.31 (m, 12H); 1.22 (d,3H).

8-((tert-Butyldiphenylsilyl)oxy)octan-2-ol

To a solution of octane-1,7-diol (1 g, 6.84 mmol) in DCM (75 mL) at 0°C. imidazole (0.94 g, 13.81 mmol) was added followed by slow addition ofa solution of tert-butyl(chloro)diphenylsilane (2.14 mL, 8.21 mmol) inDCM (using dropping funnel). The reaction allowed stir at 0° C. for 1.5h. The reaction was quenched with saturated NH₄Cl_((aq)). The aqueouslayer was extracted 3 times with a DCM (3×50 mL). The organic layer wasdried over anhydrous MgSO₄ and filtered, and the solvent was evaporated.The crude product was purified by flash silica gel column chromatography0-10% EtOAc in hexanes to obtain8-((tert-butyldiphenylsilyl)oxy)octan-2-ol (2.29 g, 5.95 mmol, 87%). ¹HNMR (300 MHz, CDCl₃) δ: ppm 7.69 (m, 4H); 7.42 (m, 6H); 3.80 (m, 1H);3.68 (t, 2H); 1.59 (m, 2H); 1.50-1.26 (m, 9H); 1.21 (d, 3H); 1.07 (s,9H).

8-((tert-Butyldiphenylsilyl)oxy)octan-2-yl decanoate

8-((tert-Butyldiphenylsilyl)oxy)octan-2-yl decanoate was synthesizedaccording to Method A. ¹H NMR (300 MHz, CDCl₃) δ: ppm 7.69 (m, 4H); 7.42(m, 6H); 4.92 (m, 1H); 3.67 (t, 2H); 2.29 (t, 2H); 1.67-1.42 (m, 6H);1.41-1.17 (m, 21H); 1.07 (s, 9H); 0.90 (m, 3H).

8-Hydroxyoctan-2-yl decanoate

To a solution of 8-[(tert-butyldiphenylsilyl)oxy]octan-2-yl decanoate(1.08 g, 2 mmol) in THF was added TBAF (8.02 mL 1 M solution in THF,8.02 mmol) and the mixture was allowed to stir at rt for 3h. The organicsolvents were evaporated under vacuum. The residue was diluted withEtOAc and washed with sat. NaHCO₃, followed by brine. The organic layerwas separated, dried over Na₂SO₄, filtered, and evaporated under vacuum.The residue was purified by silica gel chromatography with (0-40%) EtOAcin hexanes to obtain 8-hydroxyoctan-2-yl decanoate (0.55 g, 1.82 mmol,91%). ¹H NMR (300 MHz, CDCl₃) δ: ppm 4.91 (m, 1H); 3.66 (t, 2H); 2.29(t, 2H); 1.72-1.17 (m, 28H); 0.90 (m, 3H).

8-((8-(Heptadecan-9-yloxy)-8-oxooctyl)(2-hydroxyethyl)amino)octan-2-yldecanoate

Compound 98 was synthesized according to the general procedure andRepresentative Procedure 1 described above. UPLC/ELSD: RT=3.55 min. MS(ES): m/z (MH⁺) 725.0 for C₄₅H₈₉NO₅. ¹H NMR (300 MHz, CDCl₃) δ: ppm 4.89(m, 2H); 3.58 (br. m, 2H); 2.77-2.40 (m, 6H); 2.29 (m, 4H); 1.72-1.41(m, 14H); 1.28 (m, 51H); 0.90 (m, 9H).

X10. Compound 101: Heptadecan-9-yl8-((2-(4-methylpiperazin-1-yl)ethyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoateHeptadecan-9-yl8-((2-chloroethyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate

A solution of heptadecan-9-yl8-((2-hydroxyethyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate (1100 mg,1.55 mmol) in dichloromethane (25 mL) at 0° C. was addedN-Chlorosuccinimide in one portion. The reaction was allowed to stir at0° C. for 1 h followed by 1 h at room temperature. Added 90 mL ofhexanes and allowed the reaction to stir at room temperature for 20 min.Filtered off white solid through a silica gel plug and washed threetimes with hexanes. Organic layers were concentrated in vacuo. ¹H NMR(300 MHz, CDCl₃) δ: ppm 4.89 (p, 1H); 4.08 (t, 2H); 3.57 (m, 2H); 2.85(m, 2H); 2.54 (m, 4H); 2.33-2.27 (m, 4H); 1.66-1.28 (br. m, 62H); 0.90(m, 9H).

Heptadecan-9-yl8-((2-(4-methylpiperazin-1-yl)ethyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate

A solution of 1-methylpiperazine (15 mg, 0.151 mmol), heptadecan-9-yl8-((2-chloroethyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate (110 mg,0.151 mmol), K₂CO₃ (42 mg, 0.302 mmol) and KI (3 mg, 0.0151 mmol) weredissolved in 1:1 THF:MeCN (1 mL:1 mL). The reaction was allowed to stirat 65° C. for 18 hours. The reaction was cooled to room temperature,filtered and washed with hexanes and EtOAc. The organic filtrate wastransferred to separatory funnel and washed with water and brine. Driedorganic layers over Na₂SO₄, filtered and concentrated in vacuo. Theresidue was purified by silica gel chromatography [0-100% (mixture of 1%NH₄OH, 20% MeOH in dichloromethane) in dichloromethane] to obtainheptadecan-9-yl8-((2-(4-methylpiperazin-1-yl)ethyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate(36 mg, 0.045 mmol, 30%). UPLC/ELSD: RT=3.25 min. MS (ES): m/z (MH⁺)792.8 for C₄₉H₉₇N₃O₄. ¹H NMR (300 MHz, CDCl₃) δ: ppm 4.88 (p, 1H); 4.08(t, 2H); 2.57-2.45 (br. m, 20H); 2.31 (m, 3H); 1.64-1.28 (br. m, 62H);0.90 (m, 9H).

X11. Compound 103: Heptadecan-9-yl8-((2-(4-methylpiperazin-1-yl)ethyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoateHeptadecan-9-yl8-((2-chloroethyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate

To a stirred solution of heptadecan-9-yl8-((2-hydroxyethyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate (1100 mg,1.55 mmol) in dichloromethane (25 mL) at 0° C. was addedN-Chlorosuccinimide in one portion. The reaction was allowed to stir at0° C. for 1 h followed by 1h at room temperature. Added 90 mL of hexanesand allowed the reaction to stir at room temperature for 20 min.Filtered off white solid through a silica gel plug and washed threetimes with hexanes. Organic layers were concentrated in vacuo. ¹H NMR(300 MHz, CDCl₃) δ: ppm 4.89 (p, 1H); 4.08 (t, 2H); 3.57 (m, 2H); 2.85(m, 2H); 2.54 (m, 4H); 2.33-2.27 (m, 4H); 1.66-1.28 (br. m, 62H); 0.90(m, 9H).

Heptadecan-9-yl8-((2-morpholinoethyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate

A solution of morpholine (13 mg, 0.151 mmol), heptadecan-9-yl8-((2-chloroethyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate (110 mg,0.151 mmol), K₂CO₃ (42 mg, 0.302 mmol) and KI (3 mg, 0.0151 mmol) weredissolved in 1:1 THF:MeCN (1 mL:1 mL). The reaction was allowed to stirat 65° C. for 18 hours. The reaction was cooled to room temperature,filtered and washed with hexanes and EtOAc. The organic filtrate wastransferred to separatory funnel and washed with water and brine. Driedorganic layers over Na₂SO₄, filtered and concentrated in vacuo. Theresidue was purified by silica gel chromatography [0-100% (mixture of 1%NH₄OH, 20% MeOH in dichloromethane) in dichloromethane] to obtainheptadecan-9-yl8-((2-(4-methylpiperazin-1-yl)ethyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate(58 mg, 0.074 mmol, 49%). UPLC/ELSD: RT=3.53 min. MS (ES): m/z (MH⁺)779.8 for C₄₈H₉₄N₂O₅. ¹H NMR (300 MHz, CDCl₃) δ: ppm 4.86 (p, 1H); 4.05(t, 2H); 3.70 (m, 4H); 2.59-2.54 (m, 2H); 2.48-2.38 (m, 10H); 2.31-2.25(m, 4H); 1.64-1.26 (br. m, 62H); 0.88 (m, 9H).

XX9. Compound 108: Heptadecan-9-yl8-((3-acetamidopropyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate

To a solution of heptadecan-9-yl8-((3-aminopropyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate (400 mg,0.553 mmol) and triethylamine (0.15 mL, 1.10 mmol) in 10 mLdichloromethane was added dropwise at 0° C. acetyl chloride (47 μL, 0.66mmol), and the reaction mixture was allowed to warm to room temperaturefor 16 h. MS showed the product, and the mixture was diluted withdichloromethane and washed with saturated sodium bicarbonate and brine.After it was dried over sodium sulfate, the filtrate was concentratedand purified by ISCO (SiO₂: MeOH/CH₂Cl₂/1% NH₄OH 0 to 5%) to provide theproduct as a colorless oil (300 mg, 71%). LC/UV (202 nm): RT=9.14 min.MS (APCI): m/z (MH⁺) 765.7. ¹H NMR (300 MHz, CDCl₃) δ: ppm 7.41 (bs,1H); 4.85 (p, 1H, J=6.0 Hz); 4.04 (t, 2H, J=6.6 Hz); 3.40-3.25 (m, 2H);2.53-2.23 (m, 10H); 1.91 (s, 3H); 1.65-1.16 (m, 64H); 0.86 (m, 9H).

XX10. Compound 109: Heptadecan-9-yl8-((3-(methylsulfonamido)propyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate

Methanesulfonyl chloride (51 μL, 0.66 mmol) was added dropwise to a 0°C. solution of heptadecan-9-yl8-((3-aminopropyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate (400 mg,0.553 mmol) and triethylamine (0.15 mL, 1.10 mmol) in 10 mLdichloromethane, and the reaction mixture was allowed to warm to roomtemperature for 16 h. MS showed the product, and the mixture was dilutedwith dichloromethane and washed with saturated sodium bicarbonate andbrine. After drying over sodium sulfate, the filtrate was concentratedand purified by ISCO (SiO₂: MeOH/CH₂Cl₂/1% NH₄OH 0 to 5%) to provide theproduct as a colorless oil (296 mg, 88%). LC/UV (214 nm): RT=11.51 min.MS (APCI): m/z (MH⁺) 801.7. ¹H NMR (300 MHz, CDCl₃) δ: ppm 4.85 (p, 1H,J=6.0 Hz); 4.04 (t, 2H, J=6.6 Hz); 3.22 (t, 2H, J=5.8 Hz); 2.88 (s, 3H);2.53-2.23 (m, 10H); 1.73-1.16 (m, 64H); 0.87 (m, 9H).

XX11. Compound 110: Heptadecan-9-yl8-((3-(3,3-dimethylureido)propyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate

To a solution of heptadecan-9-yl8-((3-aminopropyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate (400 mg,0.553 mmol), dimethylaminopyridine (7 mg, 0.0553 mmol) and triethylamine(0.15 mL, 1.10 mmol) in 10 mL dichloromethane, dimethylcarbamic chloride(56 μL, 0.61 mmol) was added dropwise at 0° C., and the reaction mixturewas allowed to stir at room temperature for 16 h. MS showed the product.The mixture was diluted with dichloromethane and washed with saturatedsodium bicarbonate and brine. After it was dried over sodium sulfate,the filtrate was concentrated and purified by ISCO (SiO₂: MeOH/CH₂Cl₂/1%NH₄OH 0 to 5%) to afford the product as a colorless oil (267 mg, 60%).LC/UV (202 nm): RT=9.81 min. MS (APCI): m/z (MH⁺) 794.7. ¹H NMR (300MHz, CDCl₃) δ: ppm 6.13 (t, 1H, J=4.5 Hz); 4.85 (p, 1H, J=6.0 Hz); 4.04(t, 2H, J=6.6 Hz); 3.32-3.26 (m, 2H); 2.85 (s, 6H); 2.52-2.23 (m, 10H);1.67-1.18 (m, 64H); 0.87 (m, 9H).

XX12. Compound 111: Heptadecan-9-yl8-((3-(3,3-dimethylthioureido)propyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate

To a solution of heptadecan-9-yl8-((3-aminopropyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate (400 mg,0.553 mmol) and triethylamine (0.15 mL, 1.10 mmol) in 10 mLdichloromethane was added dropwise at 0° C. thiophosgene (51 μL, 0.664mmol), and the reaction mixture was allowed to stir at room temperaturefor 6 h. After this time, the reaction was cooled to 0° C., and asolution of dimethylamine in THF (2.0 M, 0.55 mL, 1.10 mmol) was added.The reaction was then allowed to stir at room temperature for 16 h. MSshowed the product, and the mixture was diluted with dichloromethane andwashed with saturated sodium bicarbonate and brine. After drying oversodium sulfate, the filtrate was concentrated and purified by ISCO(SiO₂: MeOH/CH₂Cl₂/1% NH₄OH 0 to 5%) to afford the product as a brownoil (346 mg, 77%). LC/UV (202 nm): RT=9.89 min. MS (APCI): m/z (MH⁺)810.7. ¹H NMR (300 MHz, CDCl₃) δ: ppm 8.12 (bs, 1H); 4.85 (p, 1H, J=6.0Hz); 4.04 (t, 2H, J=6.6 Hz); 3.74-3.64 (m, 2H); 3.20 (s, 6H); 2.62-2.23(m, 10H); 1.77-1.17 (m, 64H); 0.87 (m, 9H).

XX13. Compound 112: Heptadecan-9-yl8-((3-(3-methylureido)propyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate

To a 0° C. solution of heptadecan-9-yl8-((3-aminopropyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate (400 mg,0.553 mmol) in 10 mL dichloromethane was methyl isocyanate (38 mg, 0.664mmol), and the reaction mixture was allowed to stir at room temperaturefor 16 h. MS showed the product. The mixture was diluted withdichloromethane and washed with saturated sodium bicarbonate and brine.After it was dried over sodium sulfate, the filtrate was concentratedand purified by ISCO (SiO₂: MeOH/CH₂Cl₂/1% NH₄OH 0 to 5%) to afford theproduct as a colorless oil (320 mg, 70%). LC/UV (202 nm): RT=9.63 min.MS (APCI): m/z (MH⁺) 780.7. ¹H NMR (300 MHz, CDCl₃) δ: ppm 5.54 (bs,1H); 4.85 (p, 1H, J=6.0 Hz); 4.76 (bs, 1H); 4.04 (t, 2H, J=6.6 Hz); 3.23(t, 2H, J=5.8 Hz); 2.74 (d, 3H, J=2.0 Hz); 2.47 (t, 2H, J=6.0 Hz); 2.37(t, 4H, J=7.4 Hz); 2.31-2.23 (m, 4H); 1.68-1.17 (m, 64H); 0.87 (m, 9H).

XX14. Compound 113: Heptadecan-9-yl8-((3-(3-methylthioureido)propyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate

To a 0° C. solution of heptadecan-9-yl8-((3-aminopropyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate (400 mg,0.553 mmol) in 10 mL dichloromethane was added methyl isothiocyanate (45μL, 0.664 mmol), and the reaction mixture was allowed to stir at roomtemperature for 16 h. MS showed the product. The mixture wasconcentrated and purified by ISCO (SiO₂: MeOH/CH₂Cl₂/1% NH₄OH 0 to 5%)to afford the product as a colorless oil (312 mg, 70%). LC/UV (202 nm):RT=9.96 min. MS (APCI): m/z (MH⁺) 796.7. ¹H NMR (300 MHz, CDCl₃) δ: ppm4.85 (p, 1H, J=6.0 Hz); 4.04 (t, 2H, J=6.6 Hz); 3.51 (bs, 2H); 2.93 (bs,3H); 2.52 (t, 2H, J=6.0 Hz); 2.41 (t, 4H, J=7.8 Hz); 2.31-2.23 (m, 4H);1.68-1.17 (m, 66H); 0.86 (m, 9H).

XX15. Compound 114: Heptadecan-9-yl8-((3-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)propyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate

A mixture of heptadecan-9-yl8-((3-chloropropyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate (500 mg,0.67 mmol), uracil (300 mg, 2.67 mmol) and 1,8-diazabicycloundec-7-ene(150 μL, 1.07 mmol) in 3 mL DMF was heated at 100° C. in a sealed tubefor 16 h. The reaction mixture was concentrated to dryness andpartitioned between dichloromethane and water. The organic layer waswashed with brine. After it was dried over sodium sulfate, the filtratewas concentrated and purified by ISCO (SiO₂: MeOH/CH₂Cl₂/1% NH₄OH 0 to5%) to afford the product as a yellow oil (268 mg, 49%). LC/UV (202 nm):RT=8.91 min. MS (APCI): m/z (MH⁺) 818.7. ¹H NMR (300 MHz, CDCl₃) δ: ppm8.19 (bs, 1H); 7.24 (d, 1H, J=7.7 Hz); 5.64 (d, 1H, J=7.7 Hz); 4.85 (p,1H, J=6.0 Hz); 4.04 (t, 2H, J=6.6 Hz); 3.76 (t, 2H, J=7.0 Hz); 2.45-2.24(m, 10H); 1.81 (p, 2H, J=6.6 Hz); 1.68-1.17 (m, 62H); 0.87 (m, 9H).

XX16. Compound 115: Heptadecan-9-yl8-((3-(4-amino-2-oxopyrimidin-1(2H)-yl)propyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate

To a suspension of cytosine (82 mg, 0.74 mmol) in 1 mL DMF was added NaH(30 mg, 0.74 mmol) and the reaction mixture was stirred at roomtemperature for 30 min. A solution of heptadecan-9-yl8-((3-chloropropyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate (500 mg,0.67 mmol) in 2 mL DMF was then added and the mixture was heated at 100°C. in a sealed tube for 16 h. MS showed product. The reaction wasquenched with saturated sodium bicarbonate and extracted with hexanes(2λ). The combined organic layer was washed with water and brine. Afterit was dried over sodium sulfate, the filtrate was concentrated andpurified by ISCO (SiO₂: MeOH/CH₂Cl₂/1% NH₄OH 0 to 5%) to afford theproduct as a yellow oil (310 mg, 56%). LC/UV (202 nm): RT=8.32 min. MS(APCI): m/z (MH⁺) 817.7. ¹H NMR (300 MHz, CDCl₃) δ: ppm 7.34 (d, 1H,J=7.1 Hz); 5.61 (d, 1H, J=7.1 Hz); 5.44 (bs, 2H); 4.85 (p, 1H, J=6.0Hz); 4.04 (t, 2H, J=6.6 Hz); 3.79 (t, 2H, J=7.0 Hz); 2.42-2.22 (m, 9H);1.84 (t, 2H, J=6.6 Hz); 1.68-1.17 (m, 63H); 0.86 (m, 9H).

XX17. Compound 116: Heptadecan-9-yl8-((3-(6-amino-9H-purin-9-yl)propyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate

A mixture of heptadecan-9-yl8-((3-chloropropyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate (500 mg,0.67 mmol), adenine (135 mg, 1.0 mmol) and 1,8-diazabicycloundec-7-ene(137 μL, 1.0 mmol) in 2 mL DMF was heated at 90° C. in a sealed tube for16 h. The reaction mixture was concentrated to dryness and partitionedbetween dichloromethane and water. The organic layer was washed withbrine. After it was dried over sodium sulfate, the filtrate wasconcentrated and purified by ISCO (SiO₂: MeOH/CH₂Cl₂/1% NH₄OH 0 to 5%)to afford the product as a yellow oil (325 mg, 57%). LC/UV (202 nm):RT=8.47 min. MS (APCI): m/z (MH⁺) 841.7. ¹H NMR (300 MHz, CDCl₃) δ: ppm8.36 (s, 1H); 7.80 (s, 1H); 5.51 (bs, 2H); 4.85 (p, 1H, J=6.0 Hz); 4.24(t, 2H, J=7.0 Hz); 4.04 (t, 2H, J=6.6 Hz); 2.45-2.24 (m, 10H); 2.01 (p,2H, J=6.9 Hz); 1.68-1.17 (m, 62H); 0.86 (m, 9H).

XX18. Compound 118: 3,4-Dipentylphenyl8-((2-hydroxyethyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate5-Methoxy-2-(pent-1-yn-1-yl)benzaldehyde (see e.g., Bioorg. Med. Chem.Lett. 2013, 23, 1365)

A mixture of 2-bromo-5-methoxybenzaldehyde (4.30 g, 20 mmol), 1-pentyne(3.0 mL, 30 mmol), bis(triphenylphosphino)palladium chloride (702 mg, 1mmol), CuI (380 mg, 2.0 mmol) and triethylamine (5.6 mL, 40 mmol) in 60mL THF was heated to 50° C. for 16 h under nitrogen. TLC showed thedisappearance of starting material. The reaction mixture wasconcentrated to dryness. The residue was dissolved in dichloromethaneand washed with water and brine. After drying over sodium sulfate, thefiltrate was concentrated and the residue was purified by ISCO (SiO₂:EtOAc/Hexanes 0 to 5%) to afford the product as a dark brown oil (3.00g, 74%). ¹H NMR (300 MHz, CDCl₃) δ: ppm 10.49 (s, 1H); 7.42 (d, 1H,J=8.5 Hz); 7.36 (d, 1H, J=2.8 Hz); 7.07 (dd, 1H, J=8.5 Hz, 2.8 Hz); 3.84(s, 3H); 2.44 (t, 2H, J=7.0 Hz); 1.62 (m, 2H); 1.05 (t, 3H, J=7.2 Hz).

4-Methoxy-2-(pent-1-en-1-yl)-1-(pent-1-yn-1-yl)benzene

To a suspension of butyl triphenylphosphonium bromide (8.88 g, 22.2mmol) in 75 mL THF was added at 0° C. potassium tert-butoxide (2.50 g,22.2 mmol). After 30 min, a solution of5-methoxy-2-(pent-1-yn-1-yl)benzaldehyde (3.00 g, 14.8 mmol) in 25 mLTHF was then added slowly into the orange suspension. The reactionmixture was allowed to warm up to room temperature and stir for 60 h.Saturated ammonium chloride solution was added and the mixture wasextracted with ether (2λ), and the combined organic layer was washedwith brine. After drying over sodium sulfate, the filtrate wasconcentrated and the residue was purified by ISCO (SiO₂: EtOAc/Hexanes 0to 5%) to afford the product as a brown oil (3.46 g, 96%). ¹H NMR (300MHz, CDCl₃) δ: ppm 7.33 (d, 0.5H, J=8.5 Hz); 7.26 (d, 0.5H, J=8.5 Hz);6.99 (d, 0.5H, J=2.8 Hz); 6.88-6.80 (m, 1H); 6.73-6.61 (m, 1.5H); 6.25(dt, 0.5H, J=15.9 Hz, 6.9 Hz); 5.73 (dt, 0.5H, J=11.5 Hz, 7.4 Hz); 3.80(s, 3H); 2.45-2.37 (m, 2H); 2.31-2.18 (m, 2H); 1.71-1.41 (m, 4H);1.09-0.90 (m, 6H).

4-Methoxy-1,2-dipentylbenzene

A mixture of 4-methoxy-2-(pent-1-en-1-yl)-1-(pent-1-yn-1-yl)benzene(3.46 g, 14.3 mmol) and Pd/C (10%, 300 mg) in 60 mL EtOH was stirred for60 h under a hydrogen balloon. TLC showed complete reaction. Thereaction mixture was filtered through Celite and concentrated to affordthe product as a yellow oil (3.70 g, quant.), which was used for thenext step without purification. ¹H NMR (300 MHz, CDCl₃) δ: ppm 7.05 (d,1H, J=8.2 Hz); 6.72-6.55 (m, 2H); 3.78 (s, 3H); 2.59-2.50 (m, 4H);1.62-1.48 (m, 4H); 1.39-1.28 (m, 8H); 0.93-0.86 (m, 6H).

3,4-Dipentylphenol

To a solution of 4-methoxy-1,2-dipentylbenzene (3.40 g, 13.7 mmol) in 75mL dichloromethane was added dropwise at −78° C. BBr₃ (1.65 mL, 17.1mmol), and then the reaction was allowed to warm to room temperatureover 3 h. TLC showed complete reaction. The reaction was quenched byaddition of saturated sodium bicarbonate, and then it was extracted withdichloromethane (2×). The combined organic layer was washed with brineand dried over sodium sulfate. After concentration, the residue waspurified by ISCO (SiO₂: EtOAc/Hexanes 0 to 30%) to afford the product asa brown oil (3.35 g, 97%). ¹H NMR (300 MHz, CDCl₃) δ: ppm 6.99 (d, 1H,J=8.0 Hz); 6.64-6.59 (m, 2H); 4.45 (bs, 1H); 2.55-2.47 (m, 4H);1.66-1.43 (m, 4H); 1.39-1.28 (m, 8H); 0.93-0.86 (m, 6H).

3,4-Dipentylphenyl 8-bromooctanoate

To a solution of 8-bromooctanoic acid (2.23 g, 10 mmol) and3,4-dipentylphenol (2.34 g, 10 mmol) in dichloromethane (50 mL) wereadded N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (1.92g, 10 mmol) and DMAP (244 mg, 2 mmol). The reaction was allowed to stirat room temperature for 18 h. The reaction was diluted withdichloromethane and extracted with saturated sodium bicarbonate. Theorganic layer was separated and washed with brine, dried over sodiumsulfate. The organic layer was filtered and evaporated under vacuum. Theresidue was purified by ISCO (SiO₂: EtOAc/Hexanes 0 to 10%) to affordthe product as a brown oil (4.30 g, 98%). ¹H NMR (300 MHz, CDCl₃) δ: ppm7.11 (d, 1H, J=7.7 Hz); 6.84-6.77 (m, 2H); 3.41 (t, 2H, J=6.9 Hz);2.60-2.49 (m, 6H); 1.92-1.69 (m, 4H); 1.62-1.29 (m, 18H); 0.90 (m, 6H).

Nonyl 8-((2-hydroxyethyl)amino)octanoate

A mixture of nonyl 8-bromooctanoate (2.50 g, 7.15 mmol) and2-aminoethanol (4.3 mL, 71.5 mmol) in 10 mL EtOH was stirred at roomtemperature for 60 h. The reaction mixture was partitioned with hexanesand water, and the organic layer was washed with brine. After dryingover sodium sulfate, the filtrate was concentrated and purified by ISCO(SiO₂: MeOH/CH₂Cl₂/1% NH₄OH 0 to 20%) to afford the product as whitesolid (1.57 g, 66%). MS (APCI): m/z (MH⁺) 330.3. ¹H NMR (300 MHz, CDCl₃)δ: ppm 4.04 (t, 2H, J=6.6 Hz); 3.63 (t, 2H, J=5.2 Hz); 2.77 (t, 2H,J=5.1 Hz); 2.61 (t, 2H, J=7.1 Hz); 2.28 (t, 2H, J=7.4 Hz); 1.99 (bs,2H); 1.67-1.20 (m, 4H); 1.62-1.29 (m, 17H); 0.87 (m, 6H).

3,4-Dipentylphenyl8-((2-hydroxyethyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate

A solution of nonyl 8-((2-hydroxyethyl)amino)octanoate (500 mg, 1.52mmol), 3,4-dipentylphenyl 8-bromooctanoate (1.00 g, 2.27 mmol) and N,N-diisopropylethylamine (0.40 mL, 2.27 mmol) in tert-butanol (3 mL) washeated to 60° C. in a sealed tube for 60 h. The reaction was cooled toroom temperature and solvents were evaporated under vacuum. The residuewas purified by ISCO (SiO₂: MeOH/CH₂Cl₂/1% NH₄OH 0 to 5%) to obtainmixture (365 mg), and then the mixture was purified by ISCO(EtOAc/Hexanes/0.5% Et₃N 0 to 50%) to afford product as a colorless oil(80 mg). LC/UV (214 nm): RT=10.23 min. MS (APCI): m/z (MH⁺) 688.6. ¹HNMR (300 MHz, CDCl₃) δ: ppm 7.11 (d, 1H, J=8.0 Hz); 6.84-6.77 (m, 2H);4.04 (t, 2H, J=6.6 Hz); 3.51 (t, 2H, J=5.5 Hz); 2.60-2.38 (m, 12H); 2.28(t, 2H, J=7.4 Hz); 1.79-1.19 (m, 37H); 0.92-0.82 (m, 9H).

XX19. Compound 119: Nonyl8-((2-hydroxyethyl)(8-oxo-8-(4-pentylphenoxy)octyl)amino)octanoate4-Pentylphenyl 8-bromooctanoate

To a solution of 8-bromooctanoic acid (2.00 g, 8.96 mmol) and4-pentylphenol (3.07 mL g, 17.9 mmol) in dichloromethane (50 mL) wereadded N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (1.72g, 8.96 mmol) and DMAP (220 mg, 1.79 mmol). The reaction was allowed tostir at room temperature for 60 h. The reaction was diluted withdichloromethane and extracted with saturated sodium bicarbonate. Theorganic layer was separated and washed with brine, and dried over sodiumsulfate. The organic layer was filtered and evaporated under vacuum. Theresidue was purified by ISCO (SiO₂: EtOAc/Hexanes 0 to 10%) to affordthe product as a colorless oil (3.12 g, 94%). ¹H NMR (300 MHz, CDCl₃) δ:ppm 7.16 (d, 2H, J=8.5 Hz); 6.96 (d, 2H, J=8.5 Hz); 3.41 (t, 2H, J=6.9Hz); 2.61-2.49 (m, 4H); 1.92-1.69 (m, 4H); 1.65-1.25 (m, 10H); 0.88 (m,3H).

Nonyl 8-((2-hydroxyethyl)(8-oxo-8-(4-pentylphenoxy)octyl)amino)octanoate

A solution of nonyl 8-((2-hydroxyethyl)amino)octanoate (500 mg, 1.52mmol), 4-pentylphenyl 8-bromooctanoate (840 mg, 2.28 mmol) and N,N-diisopropylethylamine (0.40 mL, 2.28 mmol) in tert-butanol (3 mL) washeated to 60° C. in a sealed tube for 48 h. The reaction was cooled toroom temperature and solvents were evaporated under vacuum. The residuewas purified by ISCO (SiO₂: MeOH/CH₂Cl₂/1% NH₄OH 0 to 5%) to obtainmixture (360 mg), and then the mixture was purified by ISCO(EtOAc/Hexanes/0.5% Et₃N 0 to 50%) to afford the product as a colorlessoil (95 mg). LC/UV (214 nm): RT=9.63 min. MS (APCI): m/z (MH⁺) 618.5. ¹HNMR (300 MHz, CDCl₃) δ: ppm 7.11 (d, 1H, J=8.0 Hz); 6.84-6.77 (m, 2H);4.04 (t, 2H, J=6.6 Hz); 3.51 (t, 2H, J=5.5 Hz); 2.60-2.38 (m, 12H); 2.28(t, 2H, J=7.4 Hz); 1.79-1.19 (m, 37H); 0.92-0.82 (m, 9H).

XX20. Compound 120: Nonyl8-((2-hydroxyethyl)(8-oxo-8-(3-pentylphenoxy)octyl)amino)octanoate3-Pentylphenol (Ref: Tetrahedron Lett. 2013, 54, 52)

At −78° C., to a suspension of potassium tert-butoxide (6.73 g, 60 mmol)in 15 mL pentane were added sequentially tetramethylethylenediamine (9.0mL, 60 mmol) and BuLi (2.5 M in hexane, 24 mL, 60 mmol), and a solutionof m-cresol (2.6 mL, 25 mmol) in 10 mL pentane was added slowly. Thereaction mixture was warmed up to −20° C. for 3 h. 30 mL THF was addedand the reaction was cooled to −60° C. Butyl bromide (4.8 mL, 45 mmol)was added slowly, and the mixture was allowed warm to room temperatureand stir for 16 h. After cooled to 0° C., the reaction mixture wasacidified with 4 M HCl to pH˜3, and then extracted with ether. Thecombined organic layer was washed with brine and dried over sodiumsulfate. After concentration, the residue was purified by ISCO(EtOAc/Hexanes 0 to 5%) to provide a mixture of product with startingmaterial, which was distilled under vacuum to provide the product as acolorless oil (1.23 g, 65%). ¹H NMR (300 MHz, CDCl₃) δ: ppm 7.14 (t, 1H,J=7.7 Hz); 6.75 (d, 1H, J=7.7 Hz); 6.67-6.61 (m, 2H); 4.62 (s, 1H); 2.55(t, 2H, J=7.7 Hz); 1.67-1.52 (m, 2H); 1.38-1.24 (m, 4H); 0.88 (t, 3H,J=6.9 Hz).

3-Pentylphenyl 8-bromooctanoate

To a solution of 8-bromooctanoic acid (1.84 g, 8.20 mmol) and3-pentylphenol (1.23 g, 7.49 mmol) in dichloromethane (40 mL) were addedN-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (1.58 g,8.20 mmol) and DMAP (183 mg, 1.50 mmol). The reaction was allowed tostir at room temperature for 16 h. The reaction was diluted withdichloromethane and extracted with saturated sodium bicarbonate. Theorganic layer was separated and washed with brine, dried over sodiumsulfate. The organic layer was filtered and evaporated under vacuum. Theresidue was purified by ISCO (SiO₂: EtOAc/Hexanes 0 to 10%) to providethe product as a colorless oil (2.23 g, 80%). ¹H NMR (300 MHz, CDCl₃) δ:ppm 7.26 (t, 1H, J=8.5 Hz); 7.03 (d, 1H, J=7.6 Hz); 6.91-6.84 (m, 2H);3.41 (t, 2H, J=6.9 Hz); 2.61-2.49 (m, 4H); 1.92-1.69 (m, 4H); 1.65-1.25(m, 12H); 0.88 (t, 3H, J=6.9 Hz).

Nonyl 8-((2-hydroxyethyl)(8-oxo-8-(3-pentylphenoxy)octyl)amino)octanoate

A solution of nonyl 8-((2-hydroxyethyl)amino)octanoate (500 mg, 1.52mmol), 3-pentylphenyl 8-bromooctanoate (840 mg, 2.28 mmol) and N,N-diisopropylethylamine (0.40 mL, 2.28 mmol) in tert-butanol (3 mL) wasstirred at 60° C. in a sealed tube for 16 h. The reaction was cooled toroom temperature and solvents were evaporated under vacuum. The residuewas purified by ISCO (SiO₂: MeOH/CH₂Cl₂/1% NH₄OH 0 to 5%) to obtain amixture (247 mg), and then the mixture was purified by ISCO(EtOAc/Hexanes/0.5% Et₃N 0 to 50%) to afford the product as a colorlessoil (150 mg). LC/UV (202 nm): RT=7.45 min. MS (APCI): m/z (MH⁺) 618.5.¹H NMR (300 MHz, CDCl₃) δ: ppm 7.26 (t, 1H, J=8.5 Hz); 7.03 (d, 1H,J=7.6 Hz); 6.91-6.84 (m, 2H); 4.05 (t, 2H, J=6.6 Hz); 3.51 (t, 2H, J=5.5Hz); 2.64-2.38 (m, 10H); 2.28 (t, 2H, J=7.8 Hz); 1.79-1.19 (m, 41H);0.91-0.82 (m, 6H).

XX21. Compound 121: Heptadecan-9-yl8-((3-aminopropyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoateHeptadecan-9-yl8-((3-chloropropyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate

To a solution of heptadecan-9-yl8-((3-hydroxypropyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate (8.00 g,11.0 mmol) and triethylamine (2.0 mL, 14.4 mmol) in dichloromethane (200mL) was added dropwise methanesulfonyl chloride (1.07 mL, 13.8 mmol) at0° C., and the reaction mixture was allowed to room temperature for 16h. TLC and MS showed complete reaction. The reaction mixture was dilutedwith dichloromethane and washed with saturated sodium bicarbonate andbrine. After drying over sodium sulfate, the solvent was removed undervacuum to give the product as a brown oil (7.30 g, 89%). NMR showed thecrude contained a small amount of mesylate and desired chloride. Thiswas used for the next step without purification. MS (APCI): m/z (MH⁺)742.6. ¹H NMR (300 MHz, CDCl₃) δ: ppm 4.86 (p, 1H, J=6.0 Hz); 4.05 (t,2H, J=6.9 Hz); 3.58 (t, 2H, J=6.6 Hz); 2.58-2.22 (m, 9H); 1.92-1.16 (m,65H); 0.87 (m, 9H).

Heptadecan-9-yl8-((3-azidopropyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate

A mixture of heptadecan-9-yl8-((3-chloropropyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate (4.20 g,5.66 mmol) and sodium azide (1.75 g, 28.28 mmol) in 20 mL DMF in asealed tube was heated to 100° C. for 16 h. After it was cooled to roomtemperature, the reaction mixture was diluted with water and extractedwith hexanes. The combined organic layer was washed with water andbrine, and then dried over sodium sulfate. After filtration andconcentration, the residue was purified by ISCO (SiO₂: MeOH/CH₂Cl₂/1%NH₄OH 0 to 5%) to provide the product as a brown oil (3.66 g, 86%). MS(APCI): m/z (MH⁺) 749.7. ¹H NMR (300 MHz, CDCl₃) δ: ppm 4.85 (p, 1H,J=6.0 Hz); 4.04 (t, 2H, J=6.7 Hz); 3.32 (t, 2H, J=6.9 Hz); 2.58-2.22 (m,10H); 1.72-1.19 (m, 64H); 0.87 (m, 9H).

Heptadecan-9-yl8-((3-aminopropyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate

A mixture of heptadecan-9-yl8-((3-azidopropyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate (3.66 g, 4.89mmol) and Pd/C (10%, 400 mg) in 150 mL EtOH was stirred under hydrogenballoon for 16 h. MS showed complete reaction. The reaction mixture wasfiltered through Celite, and the filtrate was concentrated and purifiedby ISCO (SiO₂: MeOH/CH₂Cl₂/1% NH₄OH 0 to 20%) to afford the product as abrown oil (3.08 g, 87%). LC/UV (202 nm): RT=8.39 min. MS (APCI): m/z(MH⁺) 723.7. ¹H NMR (300 MHz, CDCl₃) δ: ppm 4.85 (p, 1H, J=6.0 Hz); 4.04(t, 2H, J=6.6 Hz); 2.70 (t, 2H, J=6.9 Hz); 2.46-2.24 (m, 10H); 1.65-1.16(m, 66H); 0.87 (m, 9H).

XX22. Compound 122: Heptadecan-9-yl8-((6-(decan-2-yloxy)-6-oxohexyl)(2-hydroxyethyl)amino)octanoate

Compound 122 was synthesized according to the general procedure andRepresentative Procedure 1 described above. UPLC/ELSD: RT=3.58 min. MS(ES): m/z (MH⁺) 697.1 for C₄₃H₈₅NO₅. ¹H NMR (300 MHz, CDCl₃) δ: ppm 4.91(m, 2H); 3.62 (m, 2H); 2.81-2.42 (br. m, 5H); 2.30 (m, 4H); 1.73-1.43(m, 14H); 1.28 (m, 48H); 0.90 (m, 9H).

XX23. Compound 123: Heptadecan-9-yl)8-(methyl(8-nonyloxy)-8-oxooctyl)amino)octanoate

A solution of heptadecan-9-yl 8-bromooctanoate (200 mg, 0.433 mmol) inmethanamine (10 mL, 19.92 mmol, 2M in THF) was allowed to stir at rt for18 h. The reaction mixture was concentrated in vacuo. The residue waspurified by silica gel chromatography (10-100% (mixture of 1% NH₄OH, 20%MeOH in dichloromethane) in dichloromethane) to obtain heptadecan-9-yl8-(methylamino)octanoate (113 mg, 0.27 mmol, 63%). UPLC/ELSD: RT=2.76min. MS (ES): m/z (MH⁺) 412.4 for C₂₆H₅₃NO₂. ¹H NMR (300 MHz, CDCl₃) δ:ppm 4.92 (p, 1H); 2.62 (t, 2H); 2.48 (s, 3H); 2.32-2.27 (m, 2H);1.66-1.52 (br. m, 8H); 1.28 (m, 30H); 0.90 (m, 6H).

Heptadecan-9-yl 8-(methyl(8-(nonyloxy)-8-oxooctyl)amino)octanoate

A solution of heptadecan-9-yl 8-(methylamino)octanoate (113 mg, 0.27mmol), nonyl 8-bromooctanoate (115 mg, 0.33 mmol) andN,N-diisopropylethylamine (67 μL, 0.38 mmol) and potassium iodide (5 mg,0.027 mmol) were dissolved in ethanol and was allowed to stir at 62° C.for 48 h. The reaction was cooled to rt and solvents were evaporated invacuo. The residue was purified by silica gel chromatography (0-100%(mixture of 1% NH₄OH, 20% MeOH in dichloromethane) in dichloromethane)to obtain heptadecan-9-yl8-(methyl(8-(nonyloxy)-8-oxooctyl)amino)octanoate (75 mg, 0.11 mmol,41%). UPLC/ELSD: RT=3.84 min. MS (ES): m/z (MH⁺) 681.0 for C₄₃H₈₅NO₄. ¹HNMR (300 MHz, CDCl₃) δ: ppm 4.88 (p, 1H); 4.08 (t, 2H); 2.88-2.67 (br.m, 7H); 2.34-2.27 (m, 4H); 1.80 (m, 4H); 1.63-1.52 (br. m, 10H);1.37-1.28 (br. m, 48H); 0.90 (m, 9H).

XX24. Compound 124: Di(heptadecan-9-yl)8,8′-(methylazanediyl)dioctanoate

A solution of heptadecan-9-yl 8-bromooctanoate (500 mg, 1.08 mmol) inmethanamine (11 mL, 21.67 mmol, 2M in THF) was allowed to stir at rt for6 h. The reaction mixture was concentrated in vacuo. The residue waspurified by silica gel chromatography (20-100% (mixture of 1% NH₄OH, 20%MeOH in dichloromethane) in dichloromethane) to obtaindi(heptadecan-9-yl) 8,8′-(methylazanediyl)dioctanoate (26 mg, 0.03 mmol,3%). UPLC/ELSD: RT=4.03 min. MS (ES): m/z (MH⁺) 793.3 for C₅₁H₁₀₁NO₄. ¹HNMR (300 MHz, CDCl₃) δ: ppm 4.89 (p, 2H); 2.32-2.24 (m, 11H); 1.66-1.28(br. m, 76H); 0.90 (m, 12H).

XX25. Compound 125:3-((8-(Heptadecan-9-yloxy)-8-oxooctyl)(8-(nonyloxy)-8-oxooctyl)amino)propanoicacid Heptadecan-9-yl 8-((8-(nonyloxy)-8-oxooctyl)amino)octanoate

At −78° C., to a solution of oxalyl chloride (0.25 mL, 3.0 mmol) in 3 mLdichloromethane was added dropwise a solution of DMSO (0.43 mL, 6.0mmol) in 2 mL dichloromethane, and then a solution of heptadecan-9-yl8-((3-hydroxypropyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate (1.45 g,2.0 mmol) in dichloromethane (10 mL) was added immediately. After it wasstirred for 30 min at this temperature, triethylamine (1.45 mL, 10.4mmol) was added and the reaction mixture was warmed up to roomtemperature. TLC and MS showed complete reaction (M+1: 722.7), and thereaction mixture was diluted with water and extracted with hexanes (2×).The combined organic layer was washed with brine. After drying oversodium sulfate, the filtrate was concentrated and the residue waspurified by ISCO (SiO₂: EtOAc/Hexanes/0.5% Et₃N 0 to 50%) to afford theproduct as a brown oil (810 mg, 61%). MS (APCI): m/z (MH⁺) 666.7. ¹H NMR(300 MHz, CDCl₃) δ: ppm 4.85 (p, 1H, J=6.0 Hz); 4.05 (t, 2H, J=6.9 Hz);2.56 (t, 4H, J=7.1 Hz); 2.31-2.24 (m, 4H); 1.67-1.19 (m, 63H); 0.87 (m,9H).

Heptadecan-9-yl8-((3-(benzyloxy)-3-oxopropyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate

A solution of heptadecan-9-yl8-((8-(nonyloxy)-8-oxooctyl)amino)octanoate (798 mg, 1.2 mmol) andbenzyl acrylate (293 mg, 1.8 mmol) in dichloromethane (20 mL) wasstirred at room temperature for 16 h. TLC and MS showed almost noreaction, 10 mL MeOH was added and the reaction mixture was stirred atroom temperature for 16 h. MS showed the product with a small amount ofmethyl ester (M+1: 829.8, 752.7). The reaction mixture was concentratedto dryness and purified by ISCO (SiO₂: EtOAc/hexanes 0 to 35%) to affordthe product as a colorless oil (280 mg, 28%). MS (APCI): m/z (MH⁺)829.8. ¹H NMR (300 MHz, CDCl₃) δ: ppm 7.36-7.32 (m, 5H); 5.10 (s, 2H);4.85 (p, 1H, J=6.0 Hz); 4.04 (t, 2H, J=6.9 Hz); 2.78 (t, 2H, J=6.9 Hz);2.46 (t, 2H, J=7.0 Hz); 2.36 (t, 4H, J=6.9 Hz); 2.30-2.24 (m, 4H);1.67-1.19 (m, 62H); 0.87 (m, 9H).

3-((8-(Heptadecan-9-yloxy)-8-oxooctyl)(8-(nonyloxy)-8-oxooctyl)amino)propanoicacid

A mixture of heptadecan-9-yl8-((3-(benzyloxy)-3-oxopropyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate(280 mg, 0.34 mmol) and Pd/C (10%, 28 mg) in 20 mL EtOAc was stirredunder hydrogen balloon for 1 h. MS showed complete reaction. Thereaction mixture was filtered and the filtrate was concentrated. Theresidue was purified by ISCO (SiO₂: MeOH/CH₂Cl₂ 0 to 10%) to afford theproduct as a colorless oil (230 mg, 91%). LC/UV (214 nm): RT=12.38 min.MS (APCI): m/z (MH⁺) 838.7. ¹H NMR (300 MHz, CDCl₃) δ: ppm 4.85 (p, 1H,J=6.0 Hz); 4.04 (t, 2H, J=6.6 Hz); 2.85 (t, 2H, J=6.0 Hz); 2.65 (t, 4H,J=7.7 Hz); 2.48 (t, 2H, J=6.0 Hz); 2.32-2.24 (m, 4H); 1.67-1.17 (m,63H); 0.87 (m, 9H).

XX26. Compound 126: Heptadecan-9-yl8-(methyl(4-(nonyloxy)-4-oxobutyl)amino)octanoate

A solution of heptadecan-9-yl 8-(methylamino)octanoate (103 mg, 0.25mmol), nonyl 4-bromobutanoate (88 mg, 0.30 mmol) andN,N-diisopropylethylamine (61 μL, 0.35 mmol) were dissolved in ethanoland was allowed to stir at 62° C. for 48 h. The reaction was cooled tort and solvents were evaporated in vacuo. The residue was taken-up inethyl acetate and washed with saturated aqueous sodium bicarbonate. Theorganic layer was separated and washed with brine, dried over Na₂SO₄ andevaporated in vacuo. The residue was purified by silica gelchromatography (0-100% (mixture of 1% NH₄OH, 20% MeOH indichloromethane) in dichloromethane) to obtain heptadecan-9-yl8-(methyl(4-(nonyloxy)-4-oxobutyl)amino)octanoate (90 mg, 0.14 mmol,58%). UPLC/ELSD: RT=3.58 min. MS (ES): m/z (MH⁺) 624.8 for C₃₉H₇₇NO₄. ¹HNMR (300 MHz, CDCl₃) δ: ppm 4.89 (p, 1H); 4.08 (t, 2H); 2.38-2.24 (br.m, 11H); 1.82 (m, 2H); 1.64-1.28 (br. m, 52H); 0.90 (m, 9H).

XX27. Compound 127: Nonyl8-((9-((bis(nonyloxy)phosphoryl)oxy)nonyl)(2-hydroxyethyl)amino)octanoate

Compound 127 was synthesized in the same manner as Compound 131 andaccording to the general procedure and Representative Procedure 1described above. UPLC/ELSD: RT=3.58 min. MS (ES): m/z (MH⁺) 805.1 forC₄₆H₉₄NO₇P. ¹H NMR (300 MHz, CDCl₃) δ: ppm 4.05 (m, 8H); 3.55 (m, 2H);2.59 (m, 2H); 2.46 (m, 4H); 2.31 (t, 2H), 1.67 (m, 11H); 1.29 (m, 55H);0.90 (m, 9H).

XX28. Compound 128: Heptadecan-9-yl8-((6-((1-cyclopropylnonyl)oxy)-6-oxohexyl)(2-hydroxyethyl)amino)octanoate

Compound 128 was synthesized according to the general procedure andRepresentative Procedure 1 described above. UPLC/ELSD: RT=3.67 min. MS(ES): m/z (MH⁺) 722.9 for C₄₅H₈₇NO₅. ¹H NMR (300 MHz, CDCl₃) δ: ppm 4.89(m, 1H); 4.30 (m, 1H); 3.56 (m, 2H); 2.72-2.39 (m, 6H); 2.30 (m, 4H),1.76-1.17 (m, 58H); 0.90 (m, 10H); 0.61-0.35 (m, 3H); 0.28 (m, 1H).

XX29. Compound 129: Undecyl6-((8-(dioctylamino)-8-oxooctyl)(2-hydroxyethyl)amino)hexanoate

Compound 129 was synthesized according to the general procedure andRepresentative Procedure 1 described above. UPLC/ELSD: RT=3.45 min. MS(ES): m/z (MH⁺) 695.9 for C₄₃H₈₆N₂O₄. ¹H NMR (300 MHz, CDCl₃) δ: ppm4.08 (t, 2H); 3.54 (m, 2H), 3.28 (m, 4H); 2.59 (m, 2H); 2.47 (m, 4H);2.32 (q, 4H); 1.73-1.19 (m, 58H); 0.90 (m, 9H).

XX30. Compound 130: Decan-2-yl8-((8-(dioctylamino)-8-oxooctyl)(2-hydroxyethyl)amino)octanoate

Compound 130 was synthesized according to the general procedure andRepresentative Procedure 1 described above. UPLC/ELSD: RT=3.46 min. MS(ES): m/z (MH⁺) 709.9 for C₄₄H₈₈N₂O₄. ¹H NMR (300 MHz, CDCl₃) δ: ppm4.90 (m, 1H); 3.70 (br. m, 2H), 3.35-3.15 (m, 4H); 2.96-2.41 (br. m,6H); 2.29 (m, 4H); 1.74-1.43 (m, 14H); 1.41-1.115 (m, 47H); 0.90 (m,9H).

XX31. Compound 131: Nonyl8-((7-((bis(octyloxy)phosphoryl)oxy)heptyl)(2-hydroxyethyl)amino)octanoate7-Bromoheptyl dioctyl phosphate

To a solution of POCl₃ (1.91 mL, 20.5 mmol) in DCM (20 mL) at 0° C.,Et₃N (2.85 mL, 20.4 mmol) was slowly added followed by7-bromoheptan-1-ol (4.0 g, 20.5 mmol). The reaction was allowed to stirfor 4 h at 0° C. A solution of octan-1-ol (7.10 mL, 45.11 mmol) and Et₃N(8.9 mL, 63.8 mmol) in DCM were added and the reaction was allowed tostir at rt for 16 h. The reaction was diluted with DCM and washed withsaturated NaHCO₃. The organic layer was separated, dried over Na₂SO₄,filtered, and evaporated under vacuum. The residue was purified by ISCOwith (0-30%) EtOAc in hexanes to obtain 7-bromoheptyl dioctyl phosphate(0.58 g, 1.16 mmol, 6%). ¹H NMR (300 MHz, CDCl₃) δ: ppm 4.03 (m, 6H);3.43 (t, 2H); 1.88 (m, 2H); 1.70 (m, 6H); 1.54-1.23 (m, 26H); 0.90 (m,6H).

Nonyl8-((7-((bis(octyloxy)phosphoryl)oxy)heptyl)(2-hydroxyethyl)amino)octanoate

Compound 131 was synthesized according to the general procedure andRepresentative Procedure 1 described above. UPLC/ELSD: RT=3.22 min. MS(ES): m/z (MH⁺) 750.0 for C₄₂H₈₆NO₇P. ¹H NMR (300 MHz, CDCl₃) δ: ppm4.05 (m, 8H); 3.51 (m, 2H); 2.60 (br. m, 2H); 2.46 (m, 4H); 2.31 (t,2H); 1.76-1.15 (m, 58H); 0.90 (m, 9H).

XX32. Compound 132: Decan-2-yl8-((7-((bis(octyloxy)phosphoryl)oxy)heptyl)(2-hydroxyethyl)amino)octanoate

Compound 132 was synthesized in the same manner as Compound 131 andaccording to the general procedure and Representative Procedure 1described above. UPLC/ELSD: RT=3.27 min. MS (ES): m/z (MH⁺) 764.00 forC₄₃H₈₈NO₇P. ¹H NMR (300 MHz, CDCl₃) δ: ppm 4.91 (m, 1H); 4.03 (m, 6H);3.56 (m, 2H); 2.73-2.38 (br. m, 6H); 2.29 (t, 2H); 1.79-1.16 (m, 61H);0.90 (m, 9H).

XX33. Compound 133:((2-Hydroxyethyl)azanediyl)bis(nonane-9,1-diyl)bis(2-hexyldecanoate)

Compound 133 was synthesized according to the general procedure andRepresentative Procedure 1 described above. UPLC/ELSD: RT=3.91 min. MS(ES): m/z (MH⁺) 824.0 for C₅₂H₁₀₃NO₅. ¹H NMR (300 MHz, CDCl₃) δ: ppm4.09 (t, 4H); 3.60 (m, 2H); 2.74-2.42 (br. m, 6H); 2.33 (m, 3H);1.72-1.17 (m, 76H); 0.90 (m, 12H).

XX34. Compound 134:9-((2-Hydroxyethyl)(8-(nonyloxy)-8-oxooctyl)amino)nonyl 2-hexyldecanoate

Compound 134 was synthesized according to the general procedure andRepresentative Procedure 1 described above. UPLC/ELSD: RT=3.48 min. MS(ES): m/z (MH⁺) 712.0 for C₄₄H₈₇NO₅. ¹H NMR (300 MHz, CDCl₃) δ: ppm 4.08(m, 4H); 3.55 (m, 2H); 2.67-2.39 (br. m, 6H); 2.31 (m, 3H); 1.71-1.19(m, 62H); 0.90 (m, 12H).

XX35. Compound 135:7-((8-(Decan-2-yloxy)-8-oxooctyl)(2-hydroxyethyl)amino)heptyl2-octyldecanoate

Compound 135 was synthesized according to the general procedure andRepresentative Procedure 1 described above. UPLC/ELSD: RT=3.63 min. MS(ES): m/z (MH⁺) 726.0 for C₄₅H₈₉NO₅. ¹H NMR (300 MHz, CDCl₃) δ: ppm 4.91(m, 1H); 4.08 (t, 2H); 3.57 (m, 2H); 2.73-2.40 (br. m, 6H); 2.29 (m,3H); 1.71-1.16 (m, 66H); 0.90 (m, 9H).

BA. Compound 136: Nonyl8-((2-hydroxyethyl)((9Z,12Z)-octadeca-9,12-dien-1-yl)amino)octanoateRepresentative Procedure 2 Nonyl 8-bromooctanoate (Method A)

To a solution of 8-bromooctanoic acid (5 g, 22 mmol) and nonan-1-ol(6.46 g, 45 mmol) in dichloromethane (100 mL) were addedN-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (4.3 g, 22mmol) and DMAP (547 mg, 4.5 mmol). The reaction was allowed to stir atrt for 18 h. The reaction was diluted with dichloromethane and washedwith saturated sodium bicarbonate. The organic layer was separated andwashed with brine, dried over MgSO₄. The organic layer was filtered andevaporated under vacuum. The residue was purified by silica gelchromatography (0-10% ethyl acetate in hexanes) to obtain nonyl8-bromooctanoate (6.1 g, 17 mmol, 77%).

¹H NMR (300 MHz, CDCl₃) δ: ppm 4.06 (t, 2H); 3.40 (t, 2H); 2.29 (t, 2H);1.85 (m, 2H); 1.72-0.97 (m, 22H); 0.88 (m, 3H).

Nonyl 8-((2-hydroxyethyl)amino)octanoate

A solution of nonyl 8-bromooctanoate (1.2 g, 3.4 mmol) and2-aminoethan-1-ol (5 mL, 83 mmol) in ethanol (2 mL) was allowed to stirat 62° C. for 18 h. The reaction mixture was concentrated in vacuum andthe residue was extracted with ethyl acetate and water. The organiclayer was separated and washed with water, brine and dried over Na₂SO₄.The organic layer was filtered and evaporated in vacuo. The residue waspurified by silica gel chromatography (0-100% (mixture of 1% NH₄OH, 20%MeOH in dichloromethane) in dichloromethane) to obtain nonyl8-((2-hydroxyethyl)amino)octanoate (295 mg, 0.9 mmol, 26%).

UPLC/ELSD: RT=1.29 min. MS (ES): m/z (MH⁺) 330.42 for C₁₉H₃₉NO₃

¹H NMR (300 MHz, CDCl₃) δ: ppm 4.07 (t, 2H); 3.65 (t, 2H); 2.78 (t, 2H);2.63 (t, 2H); 2.32-2.19 (m, 4H); 1.73-1.20 (m, 24H); 0.89 (m, 3H)

Nonyl8-((2-hydroxyethyl)((9Z,12Z)-octadeca-9,12-dien-1-yl)amino)octanoate

A solution of nonyl 8-((2-hydroxyethyl)amino)octanoate (150 mg, 0.46mmol), (6Z,9Z)-18-bromooctadeca-6,9-diene (165 mg, 0.5 mmol) andN,N-diisopropylethylamine (65 mg, 0.5 mmol) in ethanol (2 mL) wasallowed to stir at reflux for 48 h. The reaction was allowed to cool tort and solvents were evaporated under vacuum. The residue was purifiedby silica gel chromatography (0-10% MeOH in dichloromethane) to obtainnonyl8-((2-hydroxyethyl)((9Z,12Z)-octadeca-9,12-dien-1-yl)amino)octanoate (81mg, 0.14 mmol, 30%) as a HBr salt.

UPLC/ELSD: RT=3.24 min. MS (ES): m/z (MH⁺) 578.64 for C₃₇H₇₁NO₃

¹H NMR (300 MHz, CDCl₃) δ: ppm 10.71 (br., 1H); 5.36 (br. m, 4H); 4.04(m, 4H); 3.22-2.96 (br. m, 5H); 2.77 (m, 2H); 2.29 (m, 2H); 2.04 (br. m,4H); 1.86 (br. m, 4H); 1.66-1.17 (br. m, 40H); 0.89 (m, 6H)

BB. Compound 137: Methyl 12-(dodecyl(2-hydroxyethyl)amino)dodecanoateMethyl 12-bromododecanoate

To a solution of 12-bromododecanoic acid (2.5 g, 8.95 mmol) in THF (7mL) was added methanol (7.2 mL, 179 mmol). Sulfuric acid (0.50 mL, 8.95mmol) was added dropwise and the reaction was allowed to stir at 65° C.for two hours. The reaction mixture was washed with 5% NaHCO₃ and brine.The organic layer was dried over anhydrous Na₂SO₄, filtered, andconcentrated in vacuo. Purification by silica gel chromatography (0-20%EtOAc/hexanes) provided methyl 12-bromododecanoate (2.40 g, 92%).

¹H NMR (300 MHz, CDCl₃) δ: ppm 3.69 (s, 3H); 3.44 (t, 2H); 2.33 (t, 2H);1.88 (br. m, 2H); 1.64 (br. m, 2H); 1.45 (br. m, 2H); 1.31 (br. m, 12H).

Methyl 12-(dodecyl(2-hydroxyethyl)amino)dodecanoate

To a solution of methyl 12-((2-hydroxyethyl)amino)dodecanoate (413 mg,1.51 mmol) (isolated from the synthesis of12,12′-((2-Hydroxyethyl)azanediyl)didodecanoate) in MeCN (5 mL) wasadded 1-bromododecane (452 mg, 1.81 mmol), K₂CO₃ (418 mg, 3.02 mmol),and KI (25 mg, 0.151 mmol). The reaction was allowed to stir at 82° C.for 16 hours. The reaction mixture was cooled to room temperature,diluted with H₂O, and extracted with EtOAc. The combined organic layerswere washed with brine, dried over anhydrous Na₂SO₄, filtered, andconcentrated in vacuo. Purification by silica gel chromatography (0-100%[DCM, 20% MeOH, 1% NH₄OH]/MeOH) provided methyl12-(dodecyl(2-hydroxyethyl)amino)dodecanoate (409 mg, 61%).

UPLC/ELSD: RT=2.39 min. MS (ES): m/z (MH⁺) 442.60 for C₂₇H₅₅NO₃

¹H NMR (300 MHz, CDCl₃) δ: ppm 3.69 (s, 3H); 3.61 (t, 2H); 2.68 (t, 2H);2.54 (t, 4H); 2.32 (t, 2H); 1.64 (m, 2H); 1.50 (br. m, 4H); 1.28 (br. m,32H); 0.90 (t, 3H).

BC. Compound 138: Dinonyl 8,8′-((2-hydroxyethyl)azanediyl)dioctanoateRepresentative Procedure 3 Dinonyl8,8′-((2-hydroxyethyl)azanediyl)dioctanoate

A solution of nonyl 8-bromooctanoate (200 mg, 0.6 mmol) and2-aminoethan-1-ol (16 mg, 0.3 mmol) and N, N-diisopropylethylamine (74mg, 0.6 mmol) in THF/CH₃CN (1:1) (3 mL) was allowed to stir at 63° C.for 72 h. The reaction was cooled to rt and solvents were evaporatedunder vacuum. The residue was extracted with ethyl acetate and saturatedsodium bicarbonate. The organic layer was separated, dried over Na₂SO₄and evaporated under vacuum. The residue was purified by silica gelchromatography (0-10% MeOH in dichloromethane) to obtain dinonyl8,8′-((2-hydroxyethyl)azanediyl)dioctanoate (80 mg, 0.13 mmol, 43%).

UPLC/ELSD: RT=3.09 min. MS (ES): m/z (MH⁺) 598.85 for C₃₆H₇₁NO₅

¹H NMR (300 MHz, CDCl₃) δ: ppm 4.05 (m, 4H); 3.57 (br. m, 2H); 2.71-2.38(br. m, 6H); 2.29 (m, 4H), 1.71-1.01 (br. m, 49H), 0.88 (m, 6H).

BD. Compound 139: Di((Z)-non-2-en-1-yl)8,8′-((2-hydroxyethyl)azanediyl)dioctanoate

Compound 139 was synthesized following the Representative Procedure 3.

UPLC/ELSD: RT=2.88 min. MS (ES): m/z (MH⁺) 594.78 for C₃₆H₆₇NO₅

¹H NMR (300 MHz, CDCl₃) δ: ppm 5.60 (m, 2H); 5.50 (m, 2H); 4.59 (m, 4H);3.96 (br. m, 2H); 3.20-2.94 (br. m, 5H); 2.28 (m, 4H); 2.07 (m, 4H);1.80 (br. m 4H); 1.59 (br. m, 6H); 1.43-1.14 (br. m, 28H), 0.85 (m, 6H).

BE. Compound 140: Di((Z)-undec-2-en-1-yl)6,6′-((2-hydroxyethyl)azanediyl)dihexanoate

Compound 140 was synthesized following the Representative Procedure 3.

UPLC/ELSD: RT=2.87 min. MS (ES): m/z (MH⁺) 594.74 for C₃₆H₆₇NO₅

¹H NMR (300 MHz, CDCl₃) δ: ppm 5.73-5.44 (m, 4H); 4.62 (m, 4H); 3.55 (m,2H); 2.73-2.39 (br. m, 6H); 2.39 (m, 4H); 2.09 (m, 4H); 1.64 (m, 4H);1.55-1.14 (br. m, 33H); 0.88 (m, 6H).

BF. Compound 141: Diundecyl 6,6′-((2-hydroxyethyl)azanediyl)dihexanoate

Compound 141 was synthesized following Representative Procedure 3.

UPLC/ELSD: RT=3.03 min. MS (ES): m/z (MH⁺) 598.63 for C₃₆H₇₁NO₅

¹H NMR (300 MHz, CDCl₃) δ: ppm 4.05 (m, 4H); 3.53 (m, 2H); 2.95 (br. m,1H); 2.65-2.35 (m, 6H); 2.30 (m, 4H); 1.73-1.54 (m, 8H); 1.54-1.15 (m,40H); 0.88 (m, 6H).

BG. Compound 142: 12,12′-((2-Hydroxyethyl)azanediyl)didodecanoate12,12′-((2-Hydroxyethyl)azanediyl)didodecanoate

To a solution of methyl 12-bromododecanoate (1.5 g, 5.12 mmol) in MeCN(11 mL) was added ethanolamine (0.310 mL, 5.12 mmol), K₂CO₃ (1.42 g,10.2 mmol), and KI (85 mg, 0.512 mmol). The reaction was allowed to stirat 82° C. for 16 hours. The reaction mixture was cooled to roomtemperature, filtered, and the solids were washed with hexanes. Thefiltrate was extracted with hexanes, and the combined extracts wereconcentrated in vacuo. Purification by silica gel chromatography (0-100%[DCM, 20% MeOH, 1% NH₄OH]/MeOH) provided12,12′-((2-hydroxyethyl)azanediyl)didodecanoate (563 mg, 45%).

UPLC/ELSD: RT=1.81 min. MS (ES): m/z (MH⁺) 486.63 for C₂₈H₅₅NO₅

¹H NMR (300 MHz, CDCl₃) δ: ppm 3.69 (s, 6H); 3.59 (br. m, 2H); 2.75-2.40(br. m, 6H); 2.32 (t, 4H); 1.64 (m, 4H); 1.48 (br. m, 4H); 1.29 (br. m,28H).

BH. Compound 143: Nonyl8-((2-hydroxyethyl)(7-((2-octyldecyl)oxy)-7-oxoheptyl)amino)octanoate2-Octyldecanoic Acid

A solution of diisopropylamine (2.92 mL, 20.8 mmol) in THF (10 mL) wascooled to −78° C. and a solution of n-BuLi (7.5 mL, 18.9 mmol, 2.5 M inhexanes) was added. The reaction was allowed to warm to 0° C. To asolution of decanoic acid (2.96 g, 17.2 mmol) and NaH (754 mg, 18.9mmol, 60% w/w) in THF (20 mL) at 0° C. was added the solution of LDA andthe mixture was allowed to stir at rt for 30 min. After this time1-iodooctane (5 g, 20.8 mmol) was added and the reaction mixture washeated at 45° C. for 6 h. The reaction was quenched with 1N HCl (10 mL).The organic layer was dried over MgSO₄, filtered and evaporated undervacuum. The residue was purified by silica gel chromatography (0-20%ethyl acetate in hexanes) to yield 2-octyldecanoic acid (1.9 g, 6.6mmol).

¹H NMR (300 MHz, CDCl₃) δ: ppm 2.38 (br. m, 1H); 1.74-1.03 (br. m, 28H);0.91 (m, 6H).

2-Octyldecan-1-ol

A solution of 2-octyldecanoic acid (746 mg, 2.6 mmol) in dry THF (12 mL)was added to a stirred solution of LAH (5.2 mL, 5.2 mmol, 1M solution inTHF) in dry THF (6 mL) under nitrogen at 0° C. The reaction was allowedto warm to rt and stirred at rt for 12 h. A solution of saturatedNa₂SO₄*10H₂O solution (10 mL) was added. The solids were filteredthrough a plug of Celite. The filtrate was evaporated under vacuum andthe residue was purified by silica gel chromatography (0-20% ethylacetate in hexanes) to yield 2-octyldecan-1-ol (635 mg, 2.3 mmol).

¹H NMR (300 MHz, CDCl₃) δ: ppm 3.54 (d, 2H); 1.56-1.21 (br. m, 30H);0.91 (t, 6H).

2-Octyldecyl 7-bromoheptanoate

2-Octyldecyl 7-bromoheptanoate was synthesized according to Method A.

¹H NMR (300 MHz, CDCl₃) δ: ppm 3.96 (d, 2H); 3.40 (t, 2H); 2.31 (t, 2H);1.86 (m, 2H); 1.71-1.19 (m, 35H); 0.88 (m, 6H).

Nonyl8-((2-hydroxyethyl)(7-((2-octyldecyl)oxy)-7-oxoheptyl)amino)octanoatewas synthesized using Representative Procedure 2.

UPLC/ELSD: RT=5.23 min. MS (ES): m/z (MH⁺) 711.08 for C₄₄H₈₇NO₅

¹H NMR (300 MHz, CDCl₃) δ: ppm 4.05 (t, 2H); 3.96 (d, 2H); 3.58 (br. m,2H); 2.79-2.36 (br. m, 5H); 2.30 (m, 4H); 1.72-1.01 (br. m, 63H); 0.88(m, 9H).

BI. Compound 144: Nonyl8-((8-(dioctylamino)-8-oxooctyl)(2-hydroxyethyl)amino)octanoate8-Bromo-N,N-dioctyloctanamide

To a solution of 8-bromooctanoic acid (1 g, 2.2 mmol) and DMF (1 drop)in dichloromethane was added oxalyl chloride (0.416 mL, 2.5 mmol)dropwise. The reaction was allowed to stir for 1 h at room temperature.Solvents were evaporated and the residue was added to a solution ofdioctylamine (1.14 g, 4.8 mmol) and DMAP (100 mg, 0.8 mmol).Triethylamine was added to the reaction dropwise and the reaction wasallowed to stir for 18 h. The solvents were evaporated and the residuewas taken up in ethyl acetate and saturated NaHCO₃. The organic layerwas separated and washed with water and brine. The organic layer wasdried over Na₂SO₄, filtered and evaporated in vacuo. The residue waspurified by silica gel chromatography (0-100% (mixture of 1% NH₄OH, 20%MeOH in dichloromethane) in dichloromethane to yield a mixture of8-bromo-N,N-dioctyloctanamide and chloro-N,N-dioctyloctanamide (736 mg,1.6 mmol).

UPLC/ELSD: RT=4.02 min. MS (ES): m/z (MH⁺) 446.53 for C₂₄H₄₈BrNO

¹H NMR (300 MHz, CDCl₃) δ: ppm 3.55 (t, 0.6H); 3.42 (t, 1.4H); 3.36-3.15(m, 4H); 2.31 (t, 2H); 1.96-1.18 (m, 34H); 0.91 (m, 6H).

Nonyl 8-((8-(dioctylamino)-8-oxooctyl)(2-hydroxyethyl)amino)octanoatewas synthesized utilizing Representative Procedure 2.

UPLC/ELSD: RT=4.24 min. MS (ES): m/z (MH⁺) 696.16 for C₄₃H₈₆N₂O₄

¹H NMR (300 MHz, CDCl₃) δ: ppm 4.05 (t, 2H); 3.57 (br. m, 2H); 3.35-3.14(m, 4H); 2.80-.2.20 (m, 10H); 1.74-1.00 (br. m, 59H); 0.88 (m, 9H).

XX45. Compound 145: Heptadecan-9-yl8-((2-hydroxyethyl)(8-(methyl(octyl)amino)-8-oxooctyl)amino)octanoate

Compound 145 was synthesized according to the general procedure andRepresentative Procedure 1 described above. UPLC/ELSD: RT=2.17 min. MS(ES): m/z (MH⁺) 710.0 for C₄₄H₈₈N₂O₄. ¹H NMR (300 MHz, CDCl₃) δ: ppm4.89 (m, 1H); 3.55 (m, 2H); 3.37 (t, 1H); 3.27 (t, 1H); 2.98 (s, 1.5H);2.93 (s, 1.5H); 2.59 (m, 2H); 2.47 (m, 4H); 2.30 (m, 4H), 1.75-1.20 (m,60H); 0.90 (m, 9H).

XX46. Compound 146: Heptadecan-9-yl8-((2-hydroxyethyl)(6-(methyl(octyl)amino)-6-oxohexyl)amino)octanoate

Compound 146 was synthesized according to the general procedure andRepresentative Procedure 1 described above. UPLC/ELSD: RT=2.01 min. MS(ES): m/z (MH⁺) 682.0 for C₄₂H₈₄N₂O₄. ¹H NMR (300 MHz, CDCl₃) δ: ppm4.88 (m, 1H); 3.55 (m, 2H); 3.37 (t, 1H); 3.26 (t, 1H); 2.98 (s, 1.5H);2.93 (s, 1.5H); 2.59 (m, 2H); 2.48 (m, 4H); 2.31 (m, 4H), 1.76-1.18 (m,56H); 0.90 (m, 9H).

XX47. Compound 147: Tridecan-7-yl10-((2-hydroxyethyl)(8-(nonyloxy)-8-oxooctyl)amino)decanoate

Compound 147 was synthesized according to the general procedure andRepresentative Procedure 1 described above. UPLC/ELSD: RT=2.16 min. MS(ES): m/z (MH⁺) 683.0 for C₄₂H₈₃NO₅. ¹H NMR (300 MHz, CDCl₃) δ: ppm 4.89(m, 1H); 4.08 (m, 2H); 3.55 (m, 2H); 2.59 (m, 2H); 2.46 (m, 4H); 2.30(m, 4H), 1.72-1.18 (m, 58H); 0.90 (m, 9H).

XX48. Compound 148: Heptadecan-9-yl8-((2-hydroxyethyl)(8-((2-methoxynonyl)oxy)-8-oxooctyl)amino)octanoate1-((tert-Butyldiphenylsilyl)oxy)nonan-2-ol

TBDPSCl (8.58 g, 31.2 nmmol) was added to a mixture of nonane-1,2-diol(5.0 g, 31.2 mmol) and imidazole (4.24 g, 62.4 mmol) in DMF at RT. Thereaction was stirred at RT overnight. The reaction was diluted withwater (150 mL) and extracted with EtOAc/hexanes (1:1) (4λ). The combinedorganic layer was washed with brine, separated, dried with Na₂SO₄,filtered, and evaporated under vacuum. The residue was purified by ISCOwith (0-10%) EtOAc in hexanes to obtain1-((tert-butyldiphenylsilyl)oxy)nonan-2-ol (7.75 g, 19.4 mmol). ¹H NMR(300 MHz, DMSO) δ: ppm 7.63 (m, 4H); 7.43 (m, 6H); 4.51 (d, 1H); 3.54(m, 2H); 3.43 (m, 1H); 1.57 (m, 1H); 1.24 (m, 11H); 1.00 (s, 9H); 0.85(m, 3H).

2Methoxynonyl 8-bromnooctanoate

2-Methoxynonyl 8-bromooctanoate was synthesized following Method A inRepresentative Procedure 1. ¹H NMR (300 MHz, CDCl₃) δ: ppm 4.19 (m, 1H);4.04 (m, 1H); 3.42 (m, 6H); 2.36 (t, 2H); 1.87 (m, 2H); 1.73-1.22 (m,20H); 0.93 (m, 3H).

Heptadecan-9-yl8-((2-hydroyethyl)(8-((2-methoxynonyl)oxy)-8-oxooctyl)amino)octanoate

Compound 148 was synthesized according to the general procedure andRepresentative Procedure 1 described above. UPLC/ELSD: RT=2.48 min. MS(ES): m/z (MH⁺) 741.0 for C₄₅H₈₉NO₆. ¹H NMR (300 MHz, CDCl₃) δ: ppm 4.89(m, 1H); 4.19 (m, 1H); 4.04 (m, 1H); 3.57 (m, 2H); 3.42 (s, 3H); 3.37(m, 1H); 2.73-2.41 (m, 6H); 2.33 (m, 4H), 1.73-1.19 (m, 61H); 0.90 (m,9H).

XX49. Compound 149: Heptyl10-((8-(heptadecan-9-yloxy)-8-oxooctyl)(methyl)amino)decanoate

Compound 149 was synthesized similarly to Compound 123 and according tothe general procedure and Representative Procedure 1 described above.UPLC/ELSD: RT=2.55 min. MS (ES): m/z (MH⁺) 681.0 for C₄₃H₈₅NO₄. ¹H NMR(300 MHz, CDCl₃) δ: ppm 4.89 (m, 1H); 4.08 (t, 2H); 2.42-2.14 (m, 11H);1.73-1.17 (m, 62H); 0.90 (m, 9H).

XX50. Compound 150: Pentyl12-((8-(heptadecan-9-yloxy)-8-oxooctyl)(methyl)amino)dodecanoate

Compound 150 was synthesized similarly to Compound 123 and according tothe general procedure and Representative Procedure 1 described above.UPLC/ELSD: RT=2.47 min. MS (ES): m/z (MH⁺) 681.0 for C₄₃H₈₅NO₄. ¹H NMR(300 MHz, CDCl₃) δ: ppm 4.89 (m, 1H); 4.08 (t, 2H); 2.42-2.16 (m, 10H);1.73-1.20 (m, 63H); 0.90 (m, 9H).

XX51. Compound 151:7-((7-(Decanoyloxy)heptyl)(2-hydroxyethyl)amino)heptyl 2-octyldecanoate

Compound 151 was synthesized according to the general procedure andRepresentative Procedure 1 described above. UPLC/ELSD: RT=2.83 min. MS(ES): m/z (MH⁺) 711.0 for C₄₄H₈₇NO₅. ¹H NMR (300 MHz, CDCl₃) δ: ppm 4.07(m, 4H); 3.57 (m, 2H); 2.63 (br. m, 2H); 2.50 (m, 4H); 2.31 (m, 3H),1.71-1.19 (m, 62H); 0.90 (m, 9H).

XX52. Compound 152: Nonyl(Z)-8-((2-hydroxyethyl)(10-octyloctadec-8-en-1-yl)amino)octanoateN-Methoxy-N-methyl-2-octyldecanamide

To a solution of 2-octyl-decanoic acid (11.1 g, 39.02 mmol) and DMF(0.05 mL, 3.9 mmol) in DCM (100 mL) oxalyl chloride (3.63 mL, 42.92mmol) was added dropwise. The reaction was allowed to stir for 2 h atrt. Solvents and volatiles were evaporated under vacuum. The resultingresidue (crude 2-octyldecanoyl chloride) (11.82 g, 39.02 mmol) was takenup in DCM (100 mL) and N,O-dimethylhydroxylamine hydrochloride (4 g,40.97 mmol) and 4-dimethylaminopyridine (0.48 g, 3.9 mmol) were added.The mixture was allowed to cool to 0° C. and triethylamine (19.04 mL,136.57 mmol) was slowly added. The reaction was allowed to warm to rtand stir for 1 h. Solvents were evaporated under vacuum. The residue wasdiluted with EtOAc and washed with sat. NaHCO₃, followed by brine. Theorganic layer was separated, dried over Na₂SO₄, filtered, and evaporatedunder vacuum. The residue was purified by silica gel chromatography with(0-40%) EtOAc in hexanes to obtain N-methoxy-N-methyl-2-octyldecanamide(7.10 g, 21.68 mmol, 56%). ¹H NMR (300 MHz, CDCl₃) δ: ppm 3.70 (s, 3H);3.22 (s, 3H); 2.82 (br. m, 1H); 1.62 (m, 2H); 1.51-1.19 (m, 26H); 0.90(m, 6H).

2-Octyldecanal

A solution of N-methoxy-N-methyl-2-octyldecanamide (7.1 g, 21.68 mmol)in dry THF (2 ml) was added to a suspension of LAH (27.53 mL 1 M in THF,27.53 mmol) in dry THF (5 ml) at −45° C. The resulting suspension wasstirred for 1 h at −45° C., after which time it was allowed to warm toroom temperature and stir for 0.5 h. The reaction was cooled back to−45° C. and quenched with a sat. aqueous solution of sodium sulfatedecahydrate (2 mL). The mixture was stirred for 20 min at roomtemperature and filtered through plug of Celite. The filtrate was washedwith brine. The organic layer was separated, dried over sodium sulfate,filtered and evaporated under vacuum. The residue was purified by silicagel chromatography with (0-10%) EtOAc in hexanes to obtain2-octyldecanal (4.45 g, 16.57 mmol, 76%). ¹H NMR (300 MHz, CDCl₃) δ: ppm9.58 (d, 1H); 2.23 (m, 1H); 1.63 (m, 2H); 1.53-1.19 (m, 26H); 0.90 (m,6H).

(Z)-10-Octyloctadec-8-en-1-ol

A solution of (8-hydroxyoctyl)triphenylphosphonium bromide (3.68 g, 7.81mmol) in THF (16 mL) and HMPA was cooled in an ice bath and NaHMDS(19.52 mL 1 M, 19.52 mmol) was added. 2-Octyldecanal (1.05 g, 3.9 mmol)in THF (5 mL) was slowly added and the reaction was warmed to 30° C.After 16 h the reaction was diluted with 20 mL of water and acidifiedwith 2N HCl. The reaction was extracted with EtOAc (3×50 mL). Thecombined organic extracts were dried over sodium sulfate, filtered andconcentrated under reduce pressure. The residue was purified by silicagel chromatography (0-50%) EtOAc in hexanes to obtain(Z)-10-octyloctadec-8-en-1-ol (0.5 g, 1.30 mmol, 33%). ¹H NMR (300 MHz,CDCl₃) δ: ppm 5.24 (m, 1H); 4.90 (m, 1H); 3.53 (t, 2H); 2.14 (m, 1H);1.89 (m, 2H); 1.45 (m, 3H); 1.33-0.95 (m, 36H); 0.77 (m, 6H).

(Z)-1-Bromo-10-octyloctadec-8-ene

To a solution of PPh₃ (0.29 g, 1.11 mmol) and(8Z)-10-octyloctadec-8-en-1-ol (0.4 g, 1.05 mmol) in DCM (10 mL) at 0°C., NBS (0.22 g, 1.22 mmol) was added in one portion. The reaction wasallowed to stir at 0° C. for 1 h and then warm to rt and stir for 1 h.300 mL of hexanes were added and the mixture was filtered through asilica plug and evaporated under vacuum. 200 mL of hexanes were addedand the mixture was filtered through a silica plug and evaporated undervacuum to obtain (Z)-1-bromo-10-octyloctadec-8-ene (0.39 g, 0.88 mmol,83%). ¹H NMR (300 MHz, CDCl₃) δ: ppm 5.24 (m, 1H); 4.90 (m, 1H); 3.53(t, 2H); 2.14 (m, 1H); 1.89 (m, 2H); 1.45 (m, 3H); 1.33-0.95 (m, 36H);0.77 (m, 6H).

Nonyl (Z)-8-((2-hydroxyethyl)(10-octyloctadec-8-en-1-yl)amino)octanoate

Compound 152 was synthesized according to the general procedure andRepresentative Procedure 1 described above. UPLC/ELSD: RT=3.00 min. MS(ES): m/z (MH⁺) 694.0 for C₄₅H₈₉NO₃. ¹H NMR (300 MHz, CDCl₃) δ: ppm 5.36(m, 1H); 5.03 (m, 1H); 4.07 (t, 2H); 3.54 (t, 2H); 2.59 (t, 2H); 2.46(m, 4H); 2.30 (m, 3H); 2.01 (m, 2H); 1.63 (m, 4H); 1.53-1.03 (m, 58H);0.90 (m, 9H).

XX53. Compound 153: Nonyl8-((2-hydroxyethyl)(10-octyloctadecyl)amino)octanoate

A flask was charged with Pd(OH)₂ (20 mg) and purged with N₂. A solutionof nonyl8-[(2-hydroxyethyl)[(8Z)-10-octyloctadec-8-en-1-yl]amino]octanoate (100mg, 0.14 mmol) in EtOH (1 mL) was added. The reaction was purged with H₂and was kept under H₂ (balloon) with stirring for 16 h at rt. After thistime the reaction was purged with N₂. The reaction was filtered througha plug of Celite and washed with EtOH (50 mL). The filtrate wasevaporated under vacuum. The residue was dissolved in EtOAc and washedwith water. The organic layer was separated, dried over Na₂SO₄,filtered, and evaporated under vacuum. The residue was purified bysilica gel chromatography with (0-50%) (1%, 20% MeOH in DCM) in DCM toobtain nonyl 8-((2-hydroxyethyl)(10-octyloctadecyl)amino)octanoate(0.069 g, 0.099 mmol, 69%). UPLC/ELSD: RT=3.21 min. MS (ES): m/z (MH⁺)695.08 for C₄₅H₉₁NO₃. ¹H NMR (300 MHz, CDCl₃) δ: ppm 4.08 (t, 2H); 3.56(t, 2H); 2.62 (m, 2H); 2.48 (m, 4H); 2.31 (m, 2H); 1.64 (m, 4H);1.54-1.16 (m, 66H); 0.90 (m, 9H).

XX54. Compound 154: Heptadecan-9-yl8-((2-(2-hydroxyethoxy)ethyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate

Compound 154 was synthesized according to the general procedure andRepresentative Procedure 1 described above. UPLC/ELSD: RT=3.54 min. MS(ES): m/z (MH⁺) 755.0 for C₄₆H₉₁NO₆. ¹H NMR (300 MHz, CDCl₃) δ: ppm 4.88(m, 1H); 4.62 (m, 1H); 4.08 (t, 2H); 3.79-3.56 (m, 6H); 2.64 (m, 2H);2.47 (m, 4H); 2.31 (m, 4H), 1.73-1.20 (m, 61H); 0.90 (m, 9H).

XX55. Compound 155: tert-Butyl8-((8-(heptadecan-9-yloxy)-8-oxooctyl)(2-hydroxyethyl)amino)octanoatetert-Butyl 8-bromooctanoate

To a solution of 8-bromooctanoic acid (2 g, 8.96 mmol) in DCM (20 mL) at0° C. trifluoroacetic anhydride (2.77 mL, 19.9 mmol) was added dropwise.After 2.5 h. ^(t)BuOH (3.1 mL, 32.27 mmol) was slowly added. After 1 hthe reaction was warmed to rt and allowed to stir for 2.5 h. Thereaction was quenched with water and extracted with diethylether. Theorganic layer was separated, dried over MgSO₄, filtered, and evaporatedunder vacuum. The residue was purified by silica gel chromatography with(0-10%) EtOAc in hexanes to obtain tert-butyl 8-bromooctanoate (1.5 g,5.37 mmol, 60%). ¹H NMR (300 MHz, CDCl₃) δ: ppm 3.42 (t, 2H); 2.23 (t,2H); 1.87 (m, 2H); 1.60 (m, 2H); 1.47 (s, 11H); 1.35 (m, 4H).

tert-Butyl8-((8-(heptadecan-9-yloxy)-8-oxooctyl)(2-hydroxyethyl)amino)octanoate

Compound 155 was synthesized according to the general procedure andRepresentative Procedure 1 described above. UPLC/ELSD: RT=3.18 min. MS(ES): m/z (MH⁺) 641.0 for C₃₉H₇₇NO₅. ¹H NMR (300 MHz, CDCl₃) δ: ppm 4.89(m, 1H); 3.58 (br. m, 2H); 2.75-2.36 (br. m, 6H); 2.26 (m, 4H);1.71-1.40 (m, 22H); 1.28 (m, 35H); 0.90 (m, 6H).

XX56. Compound 156: Heptadecan-9-yl8-((1,3-dihydroxypropan-2-yl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate

Compound 156 was synthesized according to the general procedure andRepresentative Procedure 1 described above. UPLC/ELSD: RT=3.53 min. MS(ES): m/z (MH⁺) 741.0 for C₄₅H₈₉NO₆. ¹H NMR (300 MHz, CDCl₃) δ: ppm 4.88(m, 1H); 4.08 (t, 2H); 3.67 (br. m, 4H); 3.04 (m, 1H); 2.65 (m, 4H);2.32 (m, 4H), 1.72-1.44 (m, 15H); 1.28 (m, 48H); 0.90 (m, 9H).

XX57. Compound 157: Heptadecan-9-yl8-((1-hydroxypropan-2-yl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate

Compound 157 was synthesized according to the general procedure andRepresentative Procedure 1 described above. UPLC/ELSD: RT=3.56 min. MS(ES): m/z (MH⁺) 725.0 for C₄₅H₈₉NO₅. ¹H NMR (300 MHz, CDCl₃) δ: ppm 4.89(m, 1H); 4.08 (t, 2H); 3.45-3.17 (br. m, 2H); 2.94 (br. m, 1H);2.55-2.22 (m, 8H); 1.70-1.17 (m, 62H); 0.90 (m, 12H).

XX58. Compound 158: tert-Butyl8-((2-hydroxyethyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate

Compound 158 was synthesized according to the general procedure andRepresentative Procedure 1 described above. UPLC/ELSD: RT=2.23 min. MS(ES): m/z (MH⁺) 528.0 for C₃₁H₆₁NO₅. ¹H NMR (300 MHz, CDCl₃) δ: ppm 4.08(t, 2H); 3.55 (br. m, 2H); 2.60 (br. m, 2H); 2.47 (m, 4H); 2.31 (t, 2H);2.22 (t, 2H); 1.64 (br. m, 6H); 1.53-1.23 (m, 37H); 0.90 (m, 3H).

XX59. Compound 159: Heptadecan-9-yl8-((2-hydroxyethyl)(2-((2-hydroxyethyl)(8-(nonyloxy)-8-oxooctyl)amino)ethyl)amino)octanoate

Compound 159 was synthesized according to the general procedure andRepresentative Procedure 1 described above. UPLC/ELSD: RT=3.15 min. MS(ES): m/z (MH⁺) 798.0 for C₄₈H₉₆N₂O₆. ¹H NMR (300 MHz, CDCl₃) δ: ppm4.88 (m, 1H); 4.07 (t, 2H); 3.62 (br. m, 4H); 2.72-2.47 (br. m, 12H);2.31 (m, 4H); 1.72-1.42 (m, 14H); 1.28 (m, 47H); 0.90 (m, 12H).

XX60. Compound 160: 1,5-Bis(2-butylcyclopropyl)pentan-3-yl8-((2-hydroxyethyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate2-(2-Butylcyclopropyl)ethan-1-ol

2-(2-Butylcyclopropyl)ethan-1-ol was synthesized in the same manner asIntermediate C. ¹H NMR (300 MHz, CDCl₃) δ: ppm: 3.94 (t, 2H); 1.93 (m,1H); 1.59 (m, 7H); 1.39 (m, 1H); 1.12 (m, 3H); 0.90 (m, 3H); 0.00 (m,1H).

1-(2-Bromoethyl)-2-butylcyclopropane

1-(2-Bromoethyl)-2-butylcyclopropane was synthesized in the same manneras (Z)-1-Bromo-10-octyloctadec-8-ene. ¹H NMR (300 MHz, CDCl₃) δ: ppm:3.64 (t, 2H); 2.18 (m, 1H); 1.92 (m, 1H); 1.47 (m, 6H); 0.96 (m, 6H);0.00 (m, 1H).

1,5-Bis(2-butylcyclopropyl)pentan-3-ol

1,5-Bis(2-butylcyclopropyl)pentan-3-ol was synthesized in the samemanner as (5Z,12Z)-heptadeca-5,12-dien-9-ol. ¹H NMR (300 MHz, CDCl₃) δ:ppm: 3.96 (t, 1H); 1.64 (m, 21H); 1.16 (m, 6H); 0.91 (m, 6H); 0.03 (m,2H).

1,5-Bis(2-butylcyclopropyl)pentan-3-yl8-((2-hydroxyethyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate

Compound 160 was synthesized according to the general procedure andRepresentative Procedure 1 described above. UPLC/ELSD: RT=3.51 min. MS(ES): m/z (MH⁺) 735.0 for C₄₆H₈₇NO₅. ¹H NMR (300 MHz, CDCl₃) δ: ppm 4.97(m, 1H); 4.08 (t, 2H); 3.56 (br. m, 2H); 2.75-2.37 (br. m, 6H); 2.31 (m,4H); 1.74-1.05 (m, 54H); 0.92 (m, 9H); 0.67 (m, 6H); 0.31 (m, 2H).

XX61. Compound 161: Heptadecan-9-yl8-((2-hydroxyethyl)(10-(octanoyloxy)decan-2-yl)amino)octanoate10-(Benzyloxy)decan-2-ol

A solution of 10-(benzyloxy)decan-2-one (3.5 g, 13.34 mmol) in THF (10mL) was added to a stirred solution of LAH in THF (10 mL) under N2 at 0°C. The mixture was allowed to warm to rt and stir for 2 h after whichtime 10 mL of sat. Na₂SO₄.10H₂O (aq) solution was slowly added. Whitesolid precipitated. Additional solid Na₂SO₄.10H₂O was added and themixture was filtered through a plug of Celite. The filtrate was dilutedwith EtOAc and washed with brine. The organic layer was separated, driedover Na₂SO₄, filtered, and concentrated under vacuum. The residue waspurified by silica gel chromatography with (0-40%) EtOAc in hexanes toobtain 10-(benzyloxy)decan-2-ol (3.2 g, 12.1 mmol, 91%). ¹H NMR (300MHz, CDCl₃) δ: ppm 7.32 (m, 5H); 4.53 (s, 2H); 3.80 (m, 1H); 3.49 (t,2H); 1.64 (m, 2H); 1.55-1.25 (m, 132H); 1.21 (d, 3H).

9-Hydroxydecyl octanoate

9-Hydroxydecyl octanoate was synthesized following Method A. ¹H NMR (300MHz, CDCl₃) δ: ppm 4.08 (t, 2H); 3.80 (m, 1H); 2.30 (t, 2H); 1.64 (m,4H); 1.52-1.17 (m, 23H); 0.90 (m, 3H).

Heptadecan-9-yl8-((2-hydroxyethyl)(10-(octanoyloxy)decan-2-yl)amino)octanoate

Compound 161 was synthesized in a manner similar to Compound 152 andaccording to the general procedure and Representative Procedure 1described above. UPLC/ELSD: RT=3.46 min. MS (ES): m/z (MH⁺) 725.0 forC₄₅H₈₉NO₅. ¹H NMR (300 MHz, CDCl₃) δ: ppm 4.89 (m, 1H); 4.08 (t, 2H);3.49 (br. m, 2H); 2.77-2.55 (m, 2H); 2.54-2.23 (m, 7H); 1.71-1.20 (m,63H); 0.91 (m, 12H).

XX62. Compound 162:7-((2-Hydroxyethyl)(10-(octanoyloxy)decan-2-yl)amino)heptyl2-octyldecanoate

Compound 162 was synthesized according to the general procedure andRepresentative Procedure 1 described above. UPLC/ELSD: RT=3.49 min. MS(ES): m/z (MH⁺) 725.0 for C₄₅H₈₉NO₅. ¹H NMR (300 MHz, CDCl₃) δ: ppm 3.99(m, 4H); 2.72-2.48 (m, 2H); 2.48-2.17 (m, 6H); 1.55 (m, 8H); 1.44-1.10(m, 56H); 0.92-0.75 (m, 12H).

XX63. Compound 163:7-((2-Hydroxyethyl)(7-methyl-8-(nonyloxy)-8-oxooctyl)amino)heptyl2-octyldecanoate 8-Methoxyoctanoic Acid

To anhydrous MeOH (80 mL) at 0° C. KOH was added (7.54 g, 134.46 mmol)and stirred for 30 min. A solution of 8-bromooctanoic acid (10 g, 44.82mmol) in anhydrous MeOH (70 mL) was added and the resulting solution wasrefluxed for 18 h. MeOH was removed under vacuum and the residue wasacidified with 1N HCl and extracted with diethylether. The organic layerwas washed with brine, separated, dried over Na₂SO₄, filtered, andevaporated under vacuum. The residue was purified by silica gelchromatography with (0-50%) EtOAc in hexanes to obtain 8-methoxyoctanoicacid (6.3 g, 36.16 mmol, 81%). ¹H NMR (300 MHz, CDCl₃) δ: ppm 3.35 (m,5H); 2.37 (t, 2H); 1.61 (m, 4H); 1.36 (m, 6H).

8-Methoxy-2-methyloctanoic Acid

To a suspension of NaH in THF (100 mL) at 0° C., 8-methoxyoctanoic acid(5.6 g, 32.14 mmol) in THF (30 mL) was added dropwise. The reaction wasallowed to stir at rt for 30 min. The reaction was cooled to 0° C. andLDA (17.86 mL, 2M in THF, 35.71 mmol) was added dropwise. After completeaddition, the reaction was allowed to stir at 45° C. for 2 h. Thereaction was cooled to rt and methyl iodide (2.45 mL, 39.28 mmol) in THF(15 mL) was slowly added. The reaction was stirred at 45° C. for 16 h.The reaction was quenched with 1N HCl (20 mL). The quenched reaction wasevaporated under vacuum to remove volatiles. The residue was dissolvedin hexanes/EtOAc (1:1) and washed with 1N HCl (100 mL×2) followed bybrine. The organic layer was separated, dried over sodium sulfate,filtered and evaporated under vacuum. The residue was purified by silicagel chromatography with (0-15%) EtOAc in hexanes to obtain8-methoxy-2-methyloctanoic acid (3.25 g, 17.26 mmol, 54%). ¹H NMR (300MHz, CDCl₃) δ: ppm 3.35 (m, 5H); 2.49 (m, 1H); 1.70 (m, 1H); 1.59 (m,2H); 1.36 (m, 7H); 1.21 (d, 3H).

8-Hydroxy-2-methyloctanoic Acid

To a solution of 8-methoxy-2-methyloctanoic acid (1 g, 5.31 mmol) in DCM(20 mL) at −78° C., boron tribromide (13.28 mL 1 M in DCM, 13.28 mmol)was added dropwise. The reaction was allowed to warm to rt and stir atrt for 2 h. The reaction was poured into ice and extracted with DCM. Theorganic layer was separated, dried over Na₂SO₄, filtered, and evaporatedunder vacuum. The residue was purified by silica gel chromtography with(0-40%) EtOAc in hexanes to obtain 8-hydroxy-2-methyloctanoic acid (0.77g, 4.41 mmol, 83%). ¹H NMR (300 MHz, CDCl₃) δ: ppm 3.43 (t, 2H); 2.50(m, 1H); 1.94-1.64 (m, 4H); 1.56-1.26 (m, 7H); 1.20 (d, 3H).

Nonyl 8-hydroxy-2-methyloctanoate

A solution of 8-hydroxy-2-methyloctanoic acid (0.75 g, 4.31 mmol),nonan-1-ol (6.22 g, 43.1 mmol), 4-dimethylaminopyridine (0.11 g, 0.86mmol) in DCM (20 mL) under N2 was added to(3-{[(ethylimino)methylidene]amino}propyl)dimethylamine hydrochloride(0.83 g, 4.31 mmol). The reaction allowed to stir at rt for 16 h. Thereaction was diluted with DCM and washed with sat. NaHCO₃, followed bybrine. The organic layer was separated, dried over Na₂SO₄, filtered, andevaporated under vacuum. The residue was purified by silica gelchromatography with (0-20%) EtOAc in hexanes to obtain nonyl8-hydroxy-2-methyloctanoate (0.68 g, 2.26 mmol, 53%). ¹H NMR (300 MHz,CDCl₃) δ: ppm 4.08 (t, 2H); 3.42 (t, 2H); 2.45 (m, 1H); 1.87 (m, 2H);1.75-1.57 (m, 4H); 1.52-1.22 (m, 19H); 1.15 (d, 3H); 0.91 (m, 3H).

7-((2-Hydroxyethyl)(7-methyl-8-(nonyloxy)-8-oxooctyl)amino)heptyl2-octyldecanoate

Compound 163 was synthesized in a manner similar to Compound 152according to the general procedure and Representative Procedure 1described above. UPLC/ELSD: RT=3.50 min. MS (ES): m/z (MH⁺) 725.0 forC₄₅H₈₉NO₅. ¹H NMR (300 MHz, CDCl₃) δ: ppm 4.08 (t, 4H); 3.55 (m, 2H);2.67 (m, 2H); 2.53-2.24 (m, 6H); 1.72-1.10 (m, 65H); 0.90 (m, 9H).

XX64. Compound 164: Nonyl8-((8-(heptadecan-9-yloxy)-8-oxooctyl)(2-hydroxyethyl)amino)-2-methyloctanoate

Compound 164 was synthesized according to the general procedure andRepresentative Procedure 1 described above. UPLC/ELSD: RT=3.51 min. MS(ES): m/z (MH⁺) 725.0 for C₄₅H₈₉NO₅. ¹H NMR (300 MHz, CDCl₃) δ: ppm 4.89(m, 1H); 4.08 (t, 2H); 3.55 (m, 2H); 2.69-2.38 (m, 8H); 2.30 (t, 2H);1.74-1.09 (m, 65H); 0.90 (m, 9H).

XX65. Compound 165:7-((7-(Decanoyloxy)octyl)(2-hydroxyethyl)amino)heptyl 2-octyldecanoate

Compound 165 was synthesized according to the general procedure andRepresentative Procedure 1 described above. UPLC/ELSD: RT=3.55 min. MS(ES): m/z (MH⁺) 725.0 for C₄₅H₈₉NO₅. ¹H NMR (300 MHz, CDCl₃) δ: ppm 4.91(m, 1H); 4.08 (t, 2H); 3.55 (m, 2H); 2.68-2.39 (m, 8H); 2.29 (m, 3H);1.72-1.15 (m, 64H); 0.90 (m, 9H).

XX66. Compound 166:8-((8-(Heptadecan-9-yloxy)-8-oxooctyl)(2-hydroxyethyl)amino)octanoicacid

To a solution of heptadecan-9-yl8-{[8-(tert-butoxy)-8-oxooctyl](2-hydroxyethyl)amino}octanoate (0.11 g,0.17 mmol) in DCM was added trifluoroacetic acid (0.06 mL, 0.69 mmol)and the reaction was allowed to stir at rt for 40 h. Volatiles wereevaporated under vacuum. The residue was dissolved in ethylacetate andwater and extracted with ethylacetate. The organic layer was separated,dried with Na₂SO₄, filtered and concentrated under vacuum. The residuewas purified by silica gel chromatography (0-50%) (1%, 20% MeOH in DCM)in DCM to obtain8-{[8-(heptadecan-9-yloxy)-8-oxooctyl](2-hydroxyethyl)amino}octanoicacid (0.023 g, 0.04 mmol) as a colorless liquid. UPLC/ELSD: RT=2.72 min.MS (ES): m/z (MH⁺) 585.0 for C₃₅H₆₉NO₅. ¹H NMR (300 MHz, CDCl₃) δ: ppm4.87 (m, 1H); 3.98 (m, 2H); 3.25-3.05 (m, 6H); 2.32 (m, 4H); 1.82-1.45(m, 12H); 1.45-1.19 (m, 37H); 0.89 (m, 6H).

XX67. Compound 167:8-((2-Hydroxyethyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoic acid

Compound 167 was synthesized following the same procedure as Compound166. UPLC/ELSD: RT=1.57 min. MS (ES): m/z (MH+) 472.0 for C₂₇H₅₃NO₅. ¹HNMR (300 MHz, CDCl₃) δ: ppm 4.08 (m, 2H); 4.00 (m, 2H); 3.44-2.98 (m,10H); 2.35 (t, 4H); 1.85-1.55 (m, 10H); 1.33 (m, 23H); 0.90 (m, 3H).

XX68. Compound 168: Heptadecan-9-yl(Z)-8-((3-(2-cyano-3,3-dimethylguanidino)propyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate

To a solution of heptadecan-9-yl8-((3-aminopropyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate (220 mg, 0.3mmol) in 5 mL 2-propanol was added triethylamine (0.04 mL, 0.3 mmol)followed by diphenyl cyanocarbonimidate (72 mg, 0.3 mmol) and themixture stirred at rt for two hours. To the reaction mixture was added a2M dimethylamine solution in THF (0.75 mL, 1.5 mmol) and the resultingsolution heated to 75° C. for 18 hours. Additional 2M dimethylamine/THFsolution (0.75 mL, 1.5 mmol) was added and the temperature increased to85° C. After six hours the reaction was complete by LC/MS so thesolution was reduced under vacuum, diluted with DCM and washed once witha saturated aqueous sodium bicarbonate solution. The organic phase wasdried (MgSO₄), filtered and the filtrate evaporated in vacuo. Theresidue was purified by silica gel chromatography (0-50% (mixture of 1%NH₄OH, 20% MeOH in dichloromethane) in dichloromethane) to giveheptadecan-9-yl(Z)-8-((3-(2-cyano-3,3-dimethylguanidino)propyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate(119.2 mg, 0.14 mmol, 49%) as a colorless syrup. UPLC/ELSD: RT=3.52 min.MS (ES): m/z (MH⁺) 819.0 for C₄₉H₉₅N₅O₄. ¹H NMR (300 MHz, CDCl₃) δ: ppm7.62 (br. s., 1H); 4.86 (quint., 1H, J=6 Hz); 4.05 (t, 2H, J=7.5 Hz);3.68 (d, 2H, J=3 Hz); 2.99 (s, 6H); 2.59 (br. s, 2H); 2.43 (br. s, 3H);2.28 (m, 4H); 1.71 (br. s, 2H); 1.62 (m, 8H); 1.49 (m, 5H); 1.26 (br. m,50H); 0.88 (t, 9H, J=7.5 Hz).

XX69. Compound 169: Heptadecan-9-yl8-((3-((2-(dimethylamino)-3,4-dioxocyclobut-1-en-1-yl)amino)propyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate3-(Dimethylamino)-4-methoxycyclobut-3-ene-1,2-dione

To a solution of 3,4-dimethoxy-3-cyclobutene-1,2-dione (1 g, 7 mmol) in100 mL diethyl ether was added a 2M dimethylamine solution in THF (3.8mL, 7.6 mmol) and a ppt. formed almost immediately. The mixture wasstirred at rt for 24 hours and then filtered. The filter solids werewashed with diethyl ether and air-dried. The filter solids weredissolved in hot MeOH, filtered, the filtrate allowed to cool to roomtemp., then cooled to 0° C. to give a ppt. This was isolated viafiltration, washed with cold MeOH, air-dried, then dried under vacuum togive 3-(dimethylamino)-4-methoxycyclobut-3-ene-1,2-dione (0.42 g, 2.7mmol, 39%) as a pale yellow solid. ¹H NMR (300 MHz, DMSO-d6) δ: ppm 4.28(s, 3H); 3.21 (s, 3H); 3.05 (s, 3H).

Heptadecan-9-yl8-((3-((2-(dimethylamino)-3,4-dioxocyclobut-1-en-1-yl)amino)propyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate

To a solution of heptadecan-9-yl8-((3-aminopropyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate (220 mg, 0.3mmol) in 10 mL ethanol was added3-(dimethylamino)-4-methoxycyclobut-3-ene-1,2-dione (47 mg, 0.3 mmol)and the resulting colorless solution stirred at rt for 20 hours afterwhich no starting amine remained by LC/MS. The solution was concentratedin vacuo and the residue purified by silica gel chromatography (0-50%(mixture of 1% NH₄OH, 20% MeOH in dichloromethane) in dichloromethane)to give heptadecan-9-yl8-((3-((2-(dimethylamino)-3,4-dioxocyclobut-1-en-1-yl)amino)propyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate(135 mg, 0.16 mmol, 53%) as a colorless syrup. UPLC/ELSD: RT=3.51 min.MS (ES): m/z (MH⁺) 847.3 for C₅₁H₉₅N₃O₆. ¹H NMR (300 MHz, CDCl₃) δ: ppm7.86 (br. s., 1H); 4.86 (quint., 1H, J=6 Hz); 4.05 (t, 2H, J=6 Hz); 3.92(d, 2H, J=3 Hz); 3.20 (s, 6H); 2.63 (br. s, 2H); 2.42 (br. s, 3H); 2.28(m, 4H); 1.74 (br. s, 2H); 1.61 (m, 8H); 1.50 (m, 5H); 1.41 (m, 3H);1.25 (br. m, 47H); 0.88 (t, 9H, J=7.5 Hz).

XX70. Compound 170: Heptadecan-9-yl(E)-8-((3-((1-(methylamino)-2-nitrovinyl)amino)propyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate

To a solution of heptadecan-9-yl8-((3-aminopropyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate (220 mg, 0.3mmol) in 5 mL methanol was added1-methylthio-1-methylamino-2-nitroethene (45 mg, 0.3 mmol), theresulting solution heated to 70° C. and stirred for 24 hours after whichno starting amine remained by LC/MS. The solution was diluted with DCMand washed once with a saturated aqueous sodium bicarbonate solution.The organic phase was dried (MgSO₄), filtered and the filtrateevaporated in vacuo. The residue was purified by silica gelchromatography (0-50% (mixture of 1% NH₄OH, 20% MeOH in dichloromethane)in dichloromethane) to give heptadecan-9-yl(E)-8-((3-((1-(methylamino)-2-nitrovinyl)amino)propyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate(90 mg, 0.11 mmol, 36%) as a pale yellow syrup. UPLC/ELSD: RT=3.33 min.MS (ES): m/z (MH⁺) 824.3 for C₄₈H₉₄N₄O₆. ¹H NMR (300 MHz, CDCl₃) δ: ppm10.15 (d, 1H, J=9 Hz); 8.26 (d, 1H, J=27 Hz); 6.55 (d, 1H, J=9 Hz); 4.86(quint., 1H, J=6 Hz); 4.05 (t, 2H, J=6 Hz); 3.32 (br. s, 1H); 3.24 (br.s, 1H); 2.81 (dd, 3H, J=3 Hz, 12 Hz); 2.63 (br. s, 1H); 2.47 (br. s,4H); 2.28 (m, 4H); 1.77 (br. s, 2H); 1.62 (m, 5H); 1.59 (m, 6H); 1.49(m, 3H); 1.43 (m, 3H); 1.26 (br. m, 46H); 0.88 (t, 9H, J=7.5 Hz).

XX71. Compound 171: Heptadecan-9-yl8-((9-hydroxy-9-methyloctadecyl)(2-hydroxyethyl)amino)octanoate((Dec-9-en-1-yloxy)methyl)benzene

To a suspension of sodium hydride (3.88 g, 96.99 mmol) in THF (100 mL)was added 9-decen-1-ol (10 g, 63.99 mmol) slowly. After 30 min. benzylbromide (10.57 mL, 88.9 mmol) was added. The reaction was allowed tostir at rt for 18 h. The reaction was quenched with water. Solvents wereevaporated under vacuum. The residue was diluted with EtOAc and washedwith sat. NaHCO₃, followed by brine. The organic layer was separated,dried with Na₂SO₄, filtered, and evaporated under vacuum. The residuewas purified by silica gel chromatography with (0-20%) EtOAc in hexanesto obtain ((dec-9-en-1-yloxy)methyl)benzene (8.5 g, 34.5 mmol, 54%). ¹HNMR (300 MHz, CDCl₃) δ: ppm 7.32 (m, 5H); 5.83 (m, 1H); 4.98 (m, 2H);4.53 (s, 2H); 3.49 (t, 2H); 2.06 (m, 2H); 1.64 (m, 2H); 1.46-1.26 (br.m, 10H).

10-(Benzyloxy)decan-2-one

To a solution of palladium chloride (0.09 g, 0.52 mmol) and benzoquinone(3.09 g, 28.57 mmol) in DMF/Water (7:1, 12.8 mL),[(dec-9-en-1-yloxy)methyl]benzene (6.4 g, 25.98 mmol) was slowly addedand the dark brown solution was allowed to stir for 3 days at rt. Themixture was dissolved in 2N HCl (50 mL) and extracted with ether (3×50mL). The combined organic phase was washed with 2N NaOH (3×50 mL) anddried over MgSO₄. Solvents were removed under vacuum and the residue waspurified by silica gel chromatography (0-40%) ethyl acetate in hexanesto obtain 10-(benzyloxy)decan-2-one (3.44 g, 13.11 mmol, 50%). ¹H NMR(300 MHz, CDCl₃) δ: ppm 7.36 (m, 5H); 4.52 (s, 2H); 3.48 (t, 2H); 2.43(t, 2H); 2.15 (s, 3H); 1.61 (m, 4H); 1.45-1.24 (br. m, 8H).

1-(Benzyloxy)-9-methyloctadecan-9-ol

To a solution of 10-(benzyloxy)decan-2-one (1 g, 3.81 mmol) in THF (30mL) at 0° C., bromo(nonyl)magnesium (4.57 mL 1 M in diethylether, 4.57mmol) was added dropwise. The reaction was allowed to warm to rt andstir for 4 h. The reaction was quenched with water (2 mL), diethyletherwas added (200 mL) and the resulting white solid was filtered through asilica plug. The filtrate was extracted with ether. The organic layerwas washed with water, followed by brine. The organic layer wasseparated, dried over Na₂SO₄, filtered, and concentrated under vacuum.The residue was purified by silica gel chromatography with (0-40%) EtOAcin hexanes to obtain 1-(benzyloxy)-9-methyloctadecan-9-ol (0.99 g). Theproduct was impure but taken to the next step without furtherpurification.

9-Methyloctadecane-1,9-diol

Under N₂ a flask was charged with 1-(benzyloxy)-9-methyloctadecan-9-ol(1 g, 2.56 mmol), Pd(OH)₂ (100 mg) and EtOH. The reaction was purgedwith H₂ and was kept under H₂ (balloon) with stirring for 16 h at rt.The reaction was purged with N2. The reaction was filtered through aplug of Celite and the Celite was washed with EtOAc (200 mL). Thefiltrate was evaporated under vacuum. The residue was dissolved in EtOAcand was washed with water. The organic layer was separated, dried overNa₂SO₄, filtered, and evaporated under vacuum. The residue was purifiedby silica gel chromatography with EtOAc in hexanes (0-40%) to obtain9-methyloctadecane-1,9-diol (0.65 g, 2.16 mmol, 84%). ¹H NMR (300 MHz,CDCl₃) δ: ppm 3.66 (t, 2H); 1.59 (m, 2H); 1.49-1.22 (br. m, 29H); 1.17(s, 3H); 0.90 (m, 3H).

1-Bromo-9-methyloctadecan-9-ol

1-Bromo-9-methyloctadecan-9-ol was synthesized in the same manner as(Z)-1-bromo-10-octyloctadec-8-ene. ¹H NMR (300 MHz, CDCl₃) δ: ppm 3.43(t, 2H); 1.88 (m, 2H); 1.53-1.23 (br. m, 28H); 1.17 (s, 3H); 0.91 (m,3H).

Heptadecan-9-yl8-((9-hydroxy-9-methyloctadecyl)(2-hydroxyethyl)amino)octanoate

Compound 171 was synthesized according to the general procedure andRepresentative Procedure 1 described above. UPLC/ELSD: RT=3.56 min. MS(ES): m/z (MH⁺) 725.0 for C₄₆H₉₃NO₄. ¹H NMR (300 MHz, CDCl₃) δ: ppm 4.89(m, 1H); 3.55 (m, 2H); 2.60 (m, 2H); 2.47 (m, 4H); 2.30 (t, 2H);1.74-1.21 (m, 69H); 1.17 (s, 3H); 0.90 (m, 9H).

XX72. Compound 172: (R)-Decan-2-yl8-((8-(heptadecan-9-yloxy)-8-oxooctyl)(2-hydroxyethyl)amino)octanoate

Compound 172 was synthesized according to the general procedure andRepresentative Procedure 1 described above. UPLC/ELSD: RT=3.53 min. MS(ES): m/z (MH⁺) 725.0 for C₄₅H₈₉NO₅. ¹H NMR (300 MHz, CDCl₃) δ: ppm 4.91(m, 2H); 3.54 (m, 2H); 2.59 (m, 2H); 2.46 (m, 4H); 2.30 (m, 4H);1.70-1.19 (m, 66H); 0.90 (m, 9H).

XX73. Compound 173: Heptadecan-9-yl8-((3-(N-methylmethylsulfonamido)propyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate

To a solution of heptadecan-9-yl8-((3-chloropropyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate (200 mg,0.27 mmol) and N-methyl methanesulfonamide (50 uL, 0.54 mmol) in 4 mLdry DMF was added cesium carbonate (130 mg, 0.40 mmol), the resultingmixture heated to 60° C. and stirred for 24 hours, after which nostarting chloride remained by LC/MS. The mixture was allowed to cool tort, diluted with a 50% saturated aqueous sodium bicarbonate solution andextracted twice with DCM. The organics were combined, washed once withwater, dried (MgSO₄), filtered and conc. to a yellow oil. The residuewas purified by silica gel chromatography (0-50% (mixture of 1% NH₄OH,20% MeOH in dichloromethane) in dichloromethane) to give heptadecan-9-yl8-((3-(N-methylmethylsulfonamido)propyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate(85 mg, 0.11 mmol, 39%) as a pale yellow oil. UPLC/ELSD: RT=3.57 min. MS(ES): m/z (MH⁺) 816.1 for C₄₇H₉₄N₂O₆S. ¹H NMR (300 MHz, CDCl₃) δ: ppm4.86 (quint., 1H, J=6 Hz); 4.05 (t, 2H, J=6 Hz); 3.15 (t, 2H, J=7.5 Hz);2.85 (s, 3H); 2.79 (3, 3H); 2.40 (br. m, 5H); 2.28 (m, 4H); 1.72 (br. m,2H); 1.64-1.49 (m, 13H); 1.26 (br. m, 50H); 0.88 (t, 9H, J=7.5 Hz).

XX74. Compound 174: Heptadecan-9-yl8-((3-(2,5-dioxoimidazolidin-1-yl)propyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate

To a solution of heptadecan-9-yl8-((3-chloropropyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate (200 mg,0.27 mmol) and hydantoin (50 mg, 0.54 mmol) in 4 mL dry DMF was addedcesium carbonate (130 mg, 0.40 mmol), the resulting mixture heated to60° C. and stirred for 24 hours, after which no starting chlorideremained by LC/MS. The mixture was allowed to cool to rt, diluted with a50% saturated aqueous sodium bicarbonate solution and extracted twicewith DCM. The organics were combined, washed once with water, dried(MgSO₄), filtered and conc. The residue was purified by silica gelchromatography (0-50% (mixture of 1% NH₄OH, 20% MeOH in dichloromethane)in dichloromethane) to give heptadecan-9-yl8-((3-(2,5-dioxoimidazolidin-1-yl)propyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate(35 mg, 0.05 mmol, 18%) as a pale yellow oil. UPLC/ELSD: RT=3.52 min. MS(ES): m/z (MH⁺) 807.2 for C₄₈H₉₁N₃O₆. ¹H NMR (300 MHz, CDCl₃) δ: ppm5.27 (br. s, 1H); 4.86 (quint., 1H, J=6 Hz); 4.05 (t, 2H, J=6 Hz); 3.95(s, 2H); 3.55 (t, 2H, J=7.5 Hz); 2.50-2.34 (br. m, 5H); 2.26 (m, 4H);1.77 (br. s, 2H); 1.64-1.49 (m, 15H); 1.26 (br. m, 48H); 0.88 (t, 9H,J=7.5 Hz).

XX75. Compound 175: Heptadecan-9-yl8-((3-((methylcarbamoyl)oxy)propyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate

To a solution of heptadecan-9-yl8-((3-hydroxypropyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate (200 mg,0.27 mmol) and triethylamine (60 uL, 0.41 mmol) in 5 mL dry DCM at 0° C.was added methyl isocyanate (22 uL, 0.35 mmol) dropwise. The coolingbath was removed and the solution stirred at rt for 2 hours, after whichno starting alcohol remained by LC/MS. The reaction was quenched withthree drops of methanol, the mixture reduced in a stream of nitrogen andthe residue purified by silica gel chromatography (0-50% (mixture of 1%NH₄OH, 20% MeOH in dichloromethane) in dichloromethane) to giveheptadecan-9-yl8-((3-((methylcarbamoyl)oxy)propyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate(115 mg, 0.15 mmol, 53%) as a colorless oil. UPLC/ELSD: RT=3.54 min. MS(ES): m/z (MH⁺) 782.3 for C₄₇H₉₂N₂O₆. ¹H NMR (300 MHz, CDCl₃) δ: ppm4.86 (quint., 1H, J=6 Hz); 4.62 (br. s, 1H); 4.05 (m, 4H); 2.79 (d, 3H,J=3 Hz); 2.47 (br. s, 2H); 2.37 (br. m, 3H); 2.27 (m, 4H); 1.73 (br. s,2H); 1.61 (m, 7H); 1.50 (br. m, 4H); 1.40 (br. m, 4H); 1.25 (br. m,48H); 0.87 (t, 9H, J=7.5 Hz).

XX76. Compound 176: Heptadecan-9-yl8-((3-(2,5-dioxopyrrolidin-1-yl)propyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate

To a solution of heptadecan-9-yl8-((3-chloropropyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate (200 mg,0.27 mmol) and succinimide (50 mg, 0.54 mmol) in 4 mL dry DMSO was addedcesium carbonate (130 mg, 0.40 mmol), the resulting mixture heated to80° C. and stirred for 48 hours, after which no starting chlorideremained by LC/MS. The mixture was allowed to cool to rt, diluted with a50% saturated aqueous sodium bicarbonate solution and extracted threetimes with DCM. The organics were combined, washed once with water,dried (MgSO₄), filtered and conc. The residue was purified twice bysilica gel chromatography (0-50% (mixture of 1% NH₄OH, 20% MeOH indichloromethane) in dichloromethane) to giveheptadecan-9-yl8-((3-(2,5-dioxopyrrolidin-1-yl)propyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate(44 mg, 0.05 mmol, 19%) as a slightly yellow oil. UPLC/ELSD: RT=3.56min. MS (ES): m/z (MH⁺⁾806.1 for C₄₉H₉₂N₂O₆. ¹H NMR (300 MHz, CDCl₃) δ:ppm 4.86 (quint., 1H, J=6 Hz); 4.05 (t, 2H, J=6 Hz); 3.52 (t, 2H, J=7.5Hz); 2.69 (s, 4H); 2.42-2.25 (br. m, 9H); 1.71-1.58 (m, 10H); 1.50 (br.d, 4H, J=3 Hz); 1.26 (br. m, 51H); 0.88 (t, 9H, J=7.5 Hz).

XX77. Compound 177: Heptadecan-9-yl8-((3-(4-(tert-butoxymethyl)-1H-1,2,3-triazol-1-yl)propyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate

To a solution of heptadecan-9-yl8-((3-azidopropyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate (500 mg, 0.67mmol) and tert-butyl propargyl ether (100 uL, 0.73 mmol) in 4 mL THF wasadded a suspension of anhydrous copper(II) sulfate (5 mg, 0.03 mmol) andsodium ascorbate (14 mg, 0.07 mmol) in 1 mL water and the mixturestirred at rt for 24 hours, after which no starting azide remained byLC/MS. The mixture was diluted with a saturated aqueous sodiumbicarbonate solution and extracted three times with DCM. The organicswere combined, dried (MgSO₄), filtered and conc. The residue waspurified by silica gel chromatography (0-50% (mixture of 1% NH₄OH, 20%MeOH in dichloromethane) in dichloromethane) to give heptadecan-9-yl8-((3-(4-(tert-butoxymethyl)-1H-1,2,3-triazol-1-yl)propyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate(485 mg, 0.56 mmol, 84%) as a slightly yellow oil. UPLC/ELSD: RT=3.63min. MS (ES): m/z (MH⁺) 862.2 for C₅₂H₁₀₀N₄O₅. ¹H NMR (300 MHz, CDCl₃)δ: ppm 7.50 (s, 1H); 4.86 (quint., 1H, J=6 Hz); 4.59 (s, 2H); 4.36 (t,2H, J=7.5 Hz); 4.05 (t, 2H, J=6 Hz); 2.36 (br. m, 5H); 2.28 (m, 4H);2.02 (br. m, 2H); 1.62 (br. m, 8H); 1.50 (br. d, 4H, J=3 Hz); 1.28 (br.m, 60H); 0.88 (t, 9H, J=7.5 Hz).

XX78. Compound 178: Heptadecan-9-yl8-((3-(2-methoxyacetamido)propyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate

To a solution of heptadecan-9-yl8-((3-aminopropyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate (200 mg, 0.27mmol) and triethylamine (60 uL, 0.41 mmol) in 5 mL dry DCM at 0° C. wasadded methoxyacetyl chloride (30 uL, 0.33 mmol) dropwise. The coolingbath was removed and the solution stirred at rt for 24 hours, afterwhich no starting amine remained by LC/MS. The mixture was diluted witha 50% saturated aqueous sodium bicarbonate solution and extracted twicewith DCM. The organics were combined, washed once with water, dried(MgSO₄), filtered and conc. The residue was purified by silica gelchromatography (0-50% (mixture of 1% NH₄OH, 20% MeOH in dichloromethane)in dichloromethane) to give heptadecan-9-yl8-((3-(2-methoxyacetamido)propyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate(50 mg, 0.06 mmol, 23%) as a colorless oil. UPLC/ELSD: RT=3.56 min. MS(ES): m/z (MH⁺) 796.2 for C₄₈H₉₄N₂O₆. ¹H NMR (300 MHz, CDCl₃) δ: ppm7.53 (s, 1H); 4.86 (quint., 1H, J=6 Hz); 4.05 (t, 2H, J=6 Hz); 3.87 (s,2H); 3.39 (m, 5H); 2.47 (br. s, 2H); 2.36 (br. m, 3H); 2.27 (m, 4H);1.61 (m, 8H); 1.46 (br. m, 9H); 1.26 (br. m, 48H); 0.88 (t, 9H, J=7.5Hz).

XX79. Compound 179: Heptadecan-9-yl8-((3-(1H-1,2,3-triazol-1-yl)propyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoateHeptadecan-9-yl8-((8-(nonyloxy)-8-oxooctyl)(3-(4-(trimethylsilyl)-1H-1,2,3-triazol-1-yl)propyl)amino)octanoate

To a solution of heptadecan-9-yl8-((3-azidopropyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate (200 mg, 0.27mmol) and ethynyltrimethylsilane (41 uL, 0.29 mmol) in 2 mL THF wasadded a suspension of anhydrous copper(II) sulfate (2 mg, 0.01 mmol) andsodium ascorbate (5 mg, 0.02 mmol) in 0.5 mL water and the mixturestirred at rt for 20 hours, after which no starting azide remained byLC/MS. The mixture was diluted with a saturated aqueous sodiumbicarbonate solution and extracted three times with DCM. The organicswere combined, dried (MgSO₄), filtered and conc. The residue waspurified by silica gel chromatography (0-50% (mixture of 1% NH₄OH, 20%MeOH in dichloromethane) in dichloromethane) to give heptadecan-9-yl8-((8-(nonyloxy)-8-oxooctyl)(3-(4-(trimethylsilyl)-1H-1,2,3-triazol-1-yl)propyl)amino)octanoate (150 mg, 0.18 mmol,66%) as a slightly yellow oil which is a 2:1 mixture of TMS/des-TMSproduct by ¹H-NMR. Carried through as is. UPLC/ELSD: RT=3.63 min. MS(ES): m/z (MH⁺) 848.3 for C₅₀H₉₈N₄O₄Si. ¹H NMR (300 MHz, CDCl₃) δ: ppm7.55 (s, 1H); 4.86 (quint., 1H, J=6 Hz); 4.45 (t, 2H, J=7.5 Hz); 4.05(t, 2H, J=6 Hz); 3.42 (br. s, 1H); 2.28 (m, 5H); 1.65-1.45 (br. m, 14H);1.25 (br. m, 48H); 0.87 (t, 9H, J=7.5 Hz); 0.33 (s, 6H).

Heptadecan-9-yl8-((3-(1H-1,2,3-triazol-1-yl)propyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate

To a solution of (150 mg, 0.18 mmol) in 5 mL THF was added a 1Mtetrabutylammonium fluoride solution in THF (0.21 mL, 0.21 mmol) and thesolution stirred at rt for 24 hours after which the reaction hadprogressed ca. 25%. The solution was heated to 55° C. and stirred for 24hours, after which the reaction was complete by LC/MS. The solution wasdiluted with a saturated aqueous sodium bicarbonate solution andextracted twice with DCM. The organics were combined, dried (MgSO₄),filtered and conc. The residue was purified by silica gel chromatography(0-50% (mixture of 1% NH₄OH, 20% MeOH in dichloromethane) indichloromethane) to give heptadecan-9-yl8-((3-(1H-1,2,3-triazol-1-yl)propyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate(53 mg, 0.07 mmol, 39%) as a colorless oil. UPLC/ELSD: RT=3.55 min. MS(ES): m/z (MH⁺) 776.2 for C₄₇H₉₀N₄O₄. ¹H NMR (300 MHz, CDCl₃) δ: ppm7.69 (s, 1H); 7.55 (s, 1H); 4.86 (quint., 1H, J=6 Hz); 4.44 (t, 2H,J=7.5 Hz); 4.05 (t, 2H, J=6 Hz); 2.37 (br. m, 5H); 2.28 (m, 4H); 2.05(br. m, 2H); 1.61 (br. m, 8H); 1.49 (br. m, 4H); 1.26 (br. m, 51H); 0.88(t, 9H, J=7.5 Hz).

XX81. Compound 181: Heptadecan-9-yl8-((3-((methoxycarbonyl)amino)propyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate

To a solution of heptadecan-9-yl8-((3-aminopropyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate (200 mg, 0.27mmol) and triethylamine (60 uL, 0.41 mmol) in 5 mL dry DCM at 0° C. wasadded methyl chloroformate (27 uL, 0.33 mmol) dropwise. The cooling bathwas removed and the solution stirred at rt for 24 hours, after which nostarting amine remained by LC/MS. The mixture was diluted with a 50%saturated aqueous sodium bicarbonate solution and extracted twice withDCM. The organics were combined, washed once with water, dried (MgSO₄),filtered and conc. The residue was purified by silica gel chromatography(0-50% (mixture of 1% NH₄OH, 20% MeOH in dichloromethane) indichloromethane) to give heptadecan-9-yl8-((3-((methoxycarbonyl)amino)propyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate(120 mg, 0.15 mmol, 54%) as a colorless oil. UPLC/ELSD: RT=3.55 min. MS(ES): m/z (MH⁺) 782.1 for C₄₇H₉₂N₂O₆. ¹H NMR (300 MHz, CDCl₃) δ: ppm6.11 (br. s, 1H); 4.86 (quint., 1H, J=6 Hz); 4.05 (t, 2H, J=6 Hz); 3.64(s, 3H); 3.25 (br. d, 2H, J=6 Hz); 2.46 (br. s, 2H); 2.38-2.24 (m, 7H);1.61 (br. t, 9H, J=7.5 Hz); 1.50 (m, 4H); 1.42 (br. m, 3H); 1.26 (br. m,49H); 0.88 (t, 9H, J=7.5 Hz).

XX82. Compound 182: Heptadecan-9-yl8-((3-((2-(methylamino)-3,4-dioxocyclobut-1-en-1-yl)amino)propyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate3-Methoxy-4-(methylamino)cyclobut-3-ene-1,2-dione

To a solution of 3,4-dimethoxy-3-cyclobutene-1,2-dione (1 g, 7 mmol) in100 mL diethyl ether was added a 2M methylamine solution in THF (3.8 mL,7.6 mmol) and a ppt. formed almost immediately. The mixture was stirredat rt for 24 hours, then filtered, the filter solids washed with diethylether and air-dried. The filter solids were dissolved in hot EtOAc,filtered, the filtrate allowed to cool to room temp., then cooled to 0°C. to give a ppt. This was isolated via filtration, washed with coldEtOAc, air-dried, then dried under vacuum to give3-methoxy-4-(methylamino)cyclobut-3-ene-1,2-dione (0.70 g, 5 mmol, 73%)as a white solid. ¹H NMR (300 MHz, DMSO-d6) δ: ppm 8.50 (br. d, 1H, J=69Hz); 4.27 (s, 3H); 3.02 (sdd, 3H, J=42 Hz, 4.5 Hz).

Heptadecan-9-yl8-((3-((2-(methylamino)-3,4-dioxocyclobut-1-en-1-yl)amino)propyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate

To a solution of heptadecan-9-yl8-((3-aminopropyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate (200 mg, 0.28mmol) in 10 mL ethanol was added3-methoxy-4-(methylamino)cyclobut-3-ene-1,2-dione (39 mg, 0.28 mmol) andthe resulting colorless solution stirred at rt for 20 hours after whichno starting amine remained by LC/MS. The solution was concentrated invacuo and the residue purified by silica gel chromatography (0-100%(mixture of 1% NH₄OH, 20% MeOH in dichloromethane) in dichloromethane)to give heptadecan-9-yl8-((3-((2-(methylamino)-3,4-dioxocyclobut-1-en-1-yl)amino)propyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate(138 mg, 0.17 mmol, 60%) as a gummy white solid. UPLC/ELSD: RT=3. min.MS (ES): m/z (MH⁺) 833.4 for C₅₁H₉₅N₃O₆. ¹H NMR (300 MHz, CDCl₃) δ: ppm7.86 (br. s., 1H); 4.86 (quint., 1H, J=6 Hz); 4.05 (t, 2H, J=6 Hz); 3.92(d, 2H, J=3 Hz); 3.20 (s, 6H); 2.63 (br. s, 2H); 2.42 (br. s, 3H); 2.28(m, 4H); 1.74 (br. s, 2H); 1.61 (m, 8H); 1.50 (m, 5H); 1.41 (m, 3H);1.25 (br. m, 47H); 0.88 (t, 9H, J=7.5 Hz).

XX83. Compound 183: 1,3-Bis(hexyloxy)propan-2-yl8-((2-hydroxyethyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate(((1,3-Bis(hexyloxy)propan-2-yl)oxy)methyl)benzene

To a slurry of NaH (1.76 g, 43.9 mmol) in THF (40 mL) under N2 was added2-(benzyloxy)propane-1,3-diol (2 g, 10.98 mmol) and the mixture wasallowed to stir at 40° C. for 2 h. After this time 1-bromohexane (4.35g, 26.34 mmol) in DMF (2 ml) and a catalytic amount of KI were added.The reaction was refluxed for 16 h. Solvents were evaporated undervacuum. The residue was diluted with EtOAc and washed with sat. NaHCO₃,followed by brine. The organic layer was separated, dried over Na₂SO₄,filtered, and evaporated under vacuum. The residue was purified bysilica gel chromatography with (0-40%) EtOAc in hexanes to obtain(((1,3-bis(hexyloxy)propan-2-yl)oxy)methyl)benzene (1.7 g, 4.75 mmol,43%). ¹H NMR (300 MHz, CDCl₃) δ: ppm 7.34 (m, 5H); 4.73 (s, 2H); 3.75(m, 1H); 3.61-3.40 (m, 8H); 1.59 (m, 4H); 1.32 (m, 12H); 0.91 (m, 6H).

1,3-Bis(hexyloxy)propan-2-ol

1,3-Bis(hexyloxy)propan-2-ol was synthesized using the same manner as9-Methyloctadecane-1,9-diol. ¹H NMR (300 MHz, CDCl₃) δ: ppm 3.96 (m,1H); 3.48 (m, 8H); 2.37 (br. S, 1H); 1.64 (m, 2H); 1.60 (m, 4H); 1.32(m, 12H); 0.91 (m, 6H).

1,3-Bis(hexyloxy)propan-2-yl8-((2-hydroxyethyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate

Compound 183 was synthesized according to the general procedure andRepresentative Procedure 1 described above. UPLC/ELSD: RT=3.17 min. MS(ES): m/z (MH⁺) 715.0 for C₄₂H₈₃NO₇. ¹H NMR (300 MHz, CDCl₃) δ: ppm 5.15(m, 1H); 4.08 (t, 2H); 3.66-3.34 (m, 10H); 2.71-2.41 (m, 6H); 2.34 (m,4H); 1.74-1.20 (m, 50H); 0.91 (m, 9H).

XX84. Compound 184: Heptadecan-9-yl8-((2-hydroxyethyl)(8-((2-methylnonyl)oxy)-8-oxooctyl)amino)octanoate2-Methylnonyl 8-bromooctanoate

To a solution of 8-bromooctanoic acid (3.83 g, 17.18 mmol),2-methylnonan-1-ol (2.72 g, 17.18 mmol), 4-dimethylaminopyridine (0.42g, 3.44 mmol) in DCM (25 mL) under N2 was added(3-{[(ethylimino)methylidene]amino}propyl)dimethylamine hydrochloride(3.29 g, 17.18 mmol). The reaction was allowed to stir at rt for 16 h.The reaction was diluted with DCM and washed with sat. NaHCO₃, followedby brine. The organic layer was separated, dried over Na₂SO₄, filtered,and evaporated under vacuum. The residue was purified by silica gelchromatography with (0-20%) EtOAc in hexanes to obtain 2-methylnonyl8-bromooctanoate (5.1 g, 14.04 mmol, 82%). ¹H NMR (300 MHz, CDCl₃) δ:ppm 3.98 (m, 2H); 3.43 (t, 2H); 2.33 (t, 2H); 1.93-1.74 (m, 3H);1.72-1.09 (m, 20H); 0.93 (m, 6H).

Heptadecan-9-yl8-((2-hydroxyethyl)(8-((2-methylnonyl)oxy)-8-oxooctyl)amino)octanoate

Compound 184 was synthesized according to the general procedure andRepresentative Procedure 1 described above. UPLC/ELSD: RT=3.60 min. MS(ES): m/z (MH⁺) 725.0 for C₄₅H₈₉NO₅. ¹H NMR (300 MHz, CDCl₃) δ: ppm 4.89(m, 1H); 3.92 (m, 2H); 3.57 (m, 2H); 2.70-2.41 (m, 6H); 2.31 (m, 4H);1.79 (m, 1H); 1.70-1.07 (m, 60H); 0.93 (m, 12H).

XX85. Compound 185: Henicosan-11-yl6-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)hexanoate

Compound 185 was synthesized according to the general procedure andRepresentative Procedure 1 described above. UPLC/ELSD: RT=3.72 min. MS(ES): m/z (MH⁺) 739.0 for C₄₆H₉₁NO₅. ¹H NMR (300 MHz, CDCl₃) δ: ppm 4.88(m, 1H); 4.08 (t, 2H); 3.55 (m, 2H); 2.60 (m, 2H); 2.48 (m, 4H); 2.32(m, 4H); 1.72-1.41 (m, 15H); 1.28 (m, 52H); 0.90 (m, 9H).

XX86. Compound 186: Heptyl10-((2-hydroxyethyl)(10-oxo-10-(tridecan-7-yloxy)decyl)amino)decanoate

Compound 186 was synthesized according to the general procedure andRepresentative Procedure 1 described above. UPLC/ELSD: RT=3.31 min. MS(ES): m/z (MH⁺) 739.0 for C₄₂H₈₃NO₅. ¹H NMR (300 MHz, CDCl₃) δ: ppm 4.89(m, 1H); 4.08 (t, 2H); 3.55 (m, 2H); 2.58 (m, 2H); 2.47 (m, 4H); 2.30(m, 4H); 1.71-1.18 (m, 58H); 0.90 (m, 9H).

XX89. Compound 189: Heptyl10-((8-(heptadecan-9-yloxy)-8-oxooctyl)(2-hydroxyethyl)amino)decanoate

Compound 189 was synthesized according to the general procedure andRepresentative Procedure 1 described above. UPLC/ELSD: RT=3.47 min. MS(ES): m/z (MH⁺) 710.98 for C₄₄H₈₇NO₅. ¹H NMR (300 MHz, CDCl₃) δ: ppm4.89 (m, 1H); 4.08 (t, 2H); 3.55 (m, 2H); 2.61 (m, 2H); 2.47 (m, 4H);2.31 (m, 4H); 1.70-1.20 (m, 62H); 0.90 (m, 9H).

XX94. Compound 194: Heptadecan-9-yl8-((3-isobutyramidopropyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate

To a solution of heptadecan-9-yl8-((3-aminopropyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate (150 mg, 0.21mmol) and triethylamine (90 uL, 0.62 mmol) in 5 mL dry DCM at 0° C. wasadded isobutyryl chloride (35 uL, 0.31 mmol) dropwise. After 30 minutesthe cooling bath was removed and the solution stirred at rt for 90minutes, after which no starting amine remained by LC/MS. The mixturewas diluted with a 50% saturated aqueous sodium bicarbonate solution andextracted twice with DCM. The organics were combined, washed once withwater, dried (MgSO₄), filtered and conc. The residue was purified bysilica gel chromatography (0-50% (mixture of 1% NH₄OH, 20% MeOH indichloromethane) in dichloromethane) to give heptadecan-9-yl8-((3-isobutyramidopropyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate (65mg, 0.08 mmol, 39%) as a colorless oil. UPLC/ELSD: RT=3.65 min. MS (ES):m/z (MH⁺) 794.3 for C₄₉H₉₆N₂O₅. ¹H NMR (300 MHz, CDCl₃) δ: ppm 7.53 (s,1H); 4.86 (quint., 1H, J=6 Hz); 4.05 (t, 2H, J=6 Hz); 3.87 (s, 2H); 3.39(m, 5H); 2.47 (br. s, 2H); 2.36 (br. m, 3H); 2.27 (m, 4H); 1.61 (m, 8H);1.46 (br. m, 9H); 1.26 (br. m, 48H); 0.88 (t, 9H, J=7.5 Hz).

XX97. Compound 197: Heptadecan-9-yl8-((3-(2-(benzyloxy)acetamido)propyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate

To a solution of heptadecan-9-yl8-((3-aminopropyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate (300 mg, 0.41mmol) and triethylamine (145 uL, 1 mmol) in 10 mL dry DCM at 0° C. wasadded benzyloxyacetyl chloride (82 uL, 0.52 mmol) dropwise. The coolingbath was removed and the solution stirred at rt for 24 hours, afterwhich no starting amine remained by LC/MS. The mixture was diluted witha 50% saturated aqueous sodium bicarbonate solution and extracted twicewith DCM. The organics were combined, washed once with water, dried(MgSO₄), filtered and conc. The residue was purified by silica gelchromatography (0-50% (mixture of 1% NH₄OH, 20% MeOH in dichloromethane)in dichloromethane) to give heptadecan-9-yl8-((3-(2-(benzyloxy)acetamido)propyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate(179 mg, 0.21 mmol, 50%) as a colorless oil. UPLC/ELSD: RT=3.66 min. MS(ES): m/z (MH⁺) 872.4 for C₅₄H₉₈N₂O₆. ¹H NMR (300 MHz, CDCl₃) δ: ppm7.55 (s, 1H); 7.33 (m, 5H); 4.86 (quint., 1H, J=6 Hz); 4.55 (s, 2H);4.05 (t, 2H, J=6 Hz); 3.97 (s, 2H); 3.35 (quart., 2H, J=6 Hz); 2.46 (br.m, 2H); 2.28 (m, 7H); 1.65-1.48 (m, 15H); 1.26 (br. m, 50H); 0.88 (t,9H, J=7.5 Hz).

XX98. Compound 198: Heptadecan-9-yl8-((3-(2-hydroxyacetamido)propyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate

To a solution of heptadecan-9-yl8-((3-(2-(benzyloxy)acetamido)propyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate(130 mg, 0.15 mmol) in 5 mL ethanol under nitrogen was added palladium10 wt. % on carbon (approx. 20, cat.) added, the sides of the flaskwashed down with ethanol and the flask fitted with a hydrogen balloon.The flask was evacuated and back-filled with hydrogen three times, thenstirred at rt for 24 hours after which no starting ether remained byLC/MS. The flask was flushed with nitrogen, the mixture filtered throughdiatomaceous earth, the filter solids washed with ethanol and thefiltrate conc. The residue was purified by silica gel chromatography(0-50% (mixture of 1% NH₄OH, 20% MeOH in dichloromethane) indichloromethane) to give heptadecan-9-yl8-((3-(2-hydroxyacetamido)propyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate(55 mg, 0.07 mmol, 47%) as a colorless oil. UPLC/ELSD: RT=3.46 min. MS(ES): m/z (MH⁺) 782.2 for C₄₇H₉₂N₂O₆. ¹H NMR (300 MHz, CDCl₃) δ: ppm7.73 (br. s, 1H); 4.86 (quint., 1H, J=6 Hz); 4.05 (m, 4H); 3.40 (quart.,2H, J=6 Hz); 2.50 (m, 2H); 2.37 (t, 4H, J=6 Hz); 2.28 (m, 4H); 1.63 (m,8H); 1.46 (br. m, 8H); 1.26 (br. m, 49H); 0.88 (t, 9H, J=7.5 Hz).

XX100. Compound 200: Heptadecan-9-yl(E)-8-((3-(3-methyl-2-nitroguanidino)propyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoateMethyl (E/Z)—N-methyl-N′-nitrocarbamimidothioate

To a suspension of 2-methyl-1-nitro-2-thiopseudourea (1.0 g, 7.4 mmol)and cesium carbonate (2.5 g, 7.8 mmol in 8 mL dry DMF was addediodomethane (0.69 mL, 11.1 mmol) and the mixture stirred at room tempfor 24 hours. The yellow mixture was diluted with water and extractedtwice with EtOAc. The organics were combined, washed three times with a50% saturated aqueous sodium bicarbonate solution, once with brine,dried (MgSO₄), filtered and conc. to a yellow solid. This was dissolvedin hot water, the solution filtered and the filtrate cooled to 4° C. forthree days. The resulting solids were isolated via filtration, washedwith water, air-dried, then dried under vacuum to give methyl(E/Z)—N-methyl-N-nitrocarbamimidothioate (85 mg, 0.57 mmol, 8%) as apale yellow solid. ¹H NMR (300 MHz, CDCl₃) δ: ppm 10.02 (br. s, 1H);3.12 (d, 1H, J=6 Hz); 2.53 (s, 3H).

To a solution of heptadecan-9-yl8-((3-aminopropyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate (200 mg, 0.28mmol) in 5 mL methanol was added methyl(E/Z)—N-methyl-N-nitrocarbamimidothioate (45 mg, 0.3 mmol), theresulting solution heated to 70° C. and stirred for 24 hours after whichno starting amine remained by LC/MS. The solution was diluted with DCMand washed once with a saturated aqueous sodium bicarbonate solution.The organic phase was dried (MgSO₄), filtered and the filtrateevaporated in vacuo. The residue was purified by silica gelchromatography (0-50% (mixture of 1% NH₄OH, 20% MeOH in dichloromethane)in dichloromethane) to give heptadecan-9-yl(E)-8-((3-(3-methyl-2-nitroguanidino)propyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate(75 mg, 0.09 mmol, 33%) as a pale yellow syrup. UPLC/ELSD: RT=3.55 min.MS (ES): m/z (MH⁺) 825.3 for C₄₇H₉₃N₅O₆. ¹H NMR (300 MHz, CDCl₃) δ: ppm9.26 (br. s, 1H); 8.27 (br. s, 1H); 4.86 (quint., 1H, J=6 Hz); 4.05 (t,2H, J=6 Hz); 3.42 (br. s, 2H); 2.86 (d, 3H, J=6 Hz); 2.60-2.40 (br. m,5H); 2.28 (m, 4H); 1.73 (br. s, 2H); 1.65-1.40 (m, 16H); 1.26 (br. m,47H); 0.88 (t, 9H, J=7.5 Hz).

XX107. Compound 207: Heptadecan-9-yl8-((3-guanidinopropyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoateHeptadecan-9-yl6-((tert-butoxycarbonyl)amino)-2,2-dimethyl-11-(8-(nonyloxy)-8-oxooctyl)-4-oxo-3-oxa-5,7,11-triazanonadec-6-en-19-oate

To a solution of heptadecan-9-yl8-((3-aminopropyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate (300 mg, 0.41mmol) and triethylamine (230 uL, 1.66 mmol) in 10 mL dry DCM at 0° C.was added1,3-bis(tert-butoxycarbonyl)-2-(trifluoromethylsulfonyl)guanidine (325mg, 0.83 mmol) in one portion and the resulting solution allowed togradually warm to rt with stirring overnight. LC/MS showed no startingmaterial remained so the solution was diluted with DCM, washed with a50% saturated aqueous sodium bicarbonate solution, the organic layerdried (MgSO₄), filtered and conc. The residue was purified by silica gelchromatography (0-50% (mixture of 1% NH₄OH, 20% MeOH in dichloromethane)in dichloromethane) to give heptadecan-9-yl6-((tert-butoxycarbonyl)amino)-2,2-dimethyl-11-(8-(nonyloxy)-8-oxooctyl)-4-oxo-3-oxa-5,7,11-triazanonadec-6-en-19-oate(310 mg, 0.32 mmol, 77%) as a colorless oil in ca. 95% purity. Largestsingle impurity has mass corresponding to product with loss of one Bocgroup. Carried through as is. UPLC/ELSD: RT=3.90 min. MS (ES): m/z (MH⁺)966.0 for C₅₆H₁₀₈N₄O₈. ¹H NMR (300 MHz, CDCl₃) δ: ppm 11.49 (s, 1H);8.55 (br. s., 1H); 4.86 (quint., 1H, J=6 Hz); 4.05 (t, 2H, J=7.5 Hz);3.45 (quart., 2H, J=6 Hz); 2.46 (m, 2H); 2.36 (m, 4H); 2.27 (m, 4H);1.61 (m, 8H); 1.50 (m, 22H); 1.40 (m, 4H); 1.25 (br. m, 48H); 0.88 (t,9H, J=7.5 Hz).

To a solution of heptadecan-9-yl6-((tert-butoxycarbonyl)amino)-2,2-dimethyl-11-(8-(nonyloxy)-8-oxooctyl)-4-oxo-3-oxa-5,7,11-triazanonadec-6-en-19-oate(310 mg, 0.32 mmol) in 10 mL DCM was added trifluoroacetic acid (500 uL,excess) and the solution stirred at rt for 48 hours after which nostarting material remained by LC/MS. The solution was conc., the residuecodistilled with DCM twice and purified by silica gel chromatography(0-50% (mixture of 1% NH₄OH, 20% MeOH in dichloromethane) indichloromethane) to give heptadecan-9-yl8-((3-guanidinopropyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate (210 mg,0.27 mmol, 84%) as a colorless oil. UPLC/ELSD: RT=3.16 min. MS (ES): m/z(MH⁺) 766.3 for C₄₆H₉₂N₄O₄. ¹H NMR (300 MHz, CDCl₃) δ: ppm 10.92 (br. s,1H); 8.82 (br. s, 1H); 7.25 (br. s, 2H); 4.85 (quint., 1H, J=6 Hz); 4.05(t, 2H, J=6 Hz); 3.38 (br. s, 2H); 3.15 (br. s, 2H); 3.00 (br. s, 4H);2.29 (m, 4H); 2.05 (br. s, 2H); 1.91 (br. s, 3H); 1.70-1.45 (br. m,12H); 1.26 (br. m, 47H); 0.88 (t, 9H, J=7.5 Hz).

XX118. Compound 218: Heptadecan-9-yl8-((3-(4-(hydroxymethyl)-1H-1,2,3-triazol-1-yl)propyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate

To a solution of heptadecan-9-yl8-((3-(4-(tert-butoxymethyl)-1H-1,2,3-triazol-1-yl)propyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate(190 mg, 0.22 mmol) in 4 mL DCM was added trifluoroacetic acid (675 uL,excess) and the solution stirred at rt for 72 hours after which nostarting material remained by LC/MS. The solution was conc., the residuecodistilled with DCM twice and purified by silica gel chromatography(0-50% (mixture of 1% NH₄OH, 20% MeOH in dichloromethane) indichloromethane) to give heptadecan-9-yl8-((3-(4-(hydroxymethyl)-1H-1,2,3-triazol-1-yl)propyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate(113 mg, 0.14 mmol, 64%) as a colorless oil. UPLC/ELSD: RT=3.41 min. MS(ES): m/z (MH⁺) 806.1 for C₄₈H₉₂N₄O₅. ¹H NMR (300 MHz, CDCl₃) δ: ppm7.54 (s, 1H); 4.86 (quint., 1H, J=6 Hz); 4.80 (s, 2H); 4.40 (t, 2H,J=7.5 Hz); 4.05 (t, 2H, J=6 Hz); 2.38 (br. m, 5H); 2.28 (m, 5H); 2.04(br. m, 2H); 1.61 (br. m, 7H); 1.50 (br. d, 4H, J=3 Hz); 1.26 (br. m,51H); 0.88 (t, 9H, J=7.5 Hz) (hydroxyl proton not observed).

XX132. Compound 232: Nonyl8-((2-hydroxyethyl)(6-oxo-6-((4-pentylcyclohexyl)oxy)hexyl)amino)octanoate

Compound 232 was synthesized according to the general procedure andRepresentative Procedure 1 described above. UPLC/ELSD: RT=2.84 min. MS(ES): m/z (MH⁺) 596.84 for C₃₆H₆₉NO₅. ¹H NMR (300 MHz, CDCl₃) δ: ppm5.01 (m, 0.5H); 4.68 (m, 0.5H); 4.08 (t, 2H); 3.56 (m, 2H), 2.67-2.55(br. m, 2H); 2.55-2.40 (br. m, 4H); 2.31 (m, 4H); 1.97 (m, 1H); 1.82 (m,2H); 1.73-1.15 (m, 43H); 1.02 (m, 1H); 0.90 (m, 6H).

Example 2: Production of Nanoparticle Compositions A. Production ofNanoparticle Compositions

In order to investigate safe and efficacious nanoparticle compositionsfor use in the delivery of therapeutic and/or prophylactics to cells, arange of formulations are prepared and tested. Specifically, theparticular elements and ratios thereof in the lipid component ofnanoparticle compositions are optimized.

Nanoparticles can be made with mixing processes such as microfluidicsand T-junction mixing of two fluid streams, one of which contains thetherapeutic and/or prophylactic and the other has the lipid components.

Lipid compositions are prepared by combining a lipid according toFormula (I), (IA), (II), (IIa), (IIb), (IIc), (IId) or (IIe), aphospholipid (such as DOPE or DSPC, obtainable from Avanti Polar Lipids,Alabaster, Ala.), a PEG lipid (such as 1,2-dimyristoyl-sn-glycerolmethoxypolyethylene glycol, also known as PEG-DMG, obtainable fromAvanti Polar Lipids, Alabaster, Ala.), and a structural lipid (such ascholesterol, obtainable from Sigma-Aldrich, Taufkirchen, Germany, or acorticosteroid (such as prednisolone, dexamethasone, prednisone, andhydrocortisone), or a combination thereof) at concentrations of about 50mM in ethanol. Solutions should be refrigeration for storage at, forexample, −20° C. Lipids are combined to yield desired molar ratios (see,for example, Table 23) and diluted with water and ethanol to a finallipid concentration of between about 5.5 mM and about 25 mM.

Nanoparticle compositions including a therapeutic and/or prophylacticand a lipid component are prepared by combining the lipid solution witha solution including the therapeutic and/or prophylactic at lipidcomponent to therapeutic and/or prophylactic wt:wt ratios between about5:1 and about 50:1. The lipid solution is rapidly injected using aNanoAssemblr microfluidic based system at flow rates between about 10ml/min and about 18 ml/min into the therapeutic and/or prophylacticsolution to produce a suspension with a water to ethanol ratio betweenabout 1:1 and about 4:1.

For nanoparticle compositions including an RNA, solutions of the RNA atconcentrations of 0.1 mg/ml in deionized water are diluted in 50 mMsodium citrate buffer at a pH between 3 and 4 to form a stock solution.

Nanoparticle compositions can be processed by dialysis to remove ethanoland achieve buffer exchange. Formulations are dialyzed twice againstphosphate buffered saline (PBS), pH 7.4, at volumes 200 times that ofthe primary product using Slide-A-Lyzer cassettes (Thermo FisherScientific Inc., Rockford, Ill.) with a molecular weight cutoff of 10kD. The first dialysis is carried out at room temperature for 3 hours.The formulations are then dialyzed overnight at 40° C. The resultingnanoparticle suspension is filtered through 0.2 μm sterile filters(Sarstedt, Nümbrecht, Germany) into glass vials and sealed with crimpclosures. Nanoparticle composition solutions of 0.01 mg/ml to 0.10 mg/mlare generally obtained.

The method described above induces nano-precipitation and particleformation. Alternative processes including, but not limited to,T-junction and direct injection, may be used to achieve the samenano-precipitation.

B. Characterization of Nanoparticle Compositions

A Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, Worcestershire,UK) can be used to determine the particle size, the polydispersity index(PDI) and the zeta potential of the nanoparticle compositions in 1×PBSin determining particle size and 15 mM PBS in determining zetapotential.

Ultraviolet-visible spectroscopy can be used to determine theconcentration of a therapeutic and/or prophylactic (e.g., RNA) innanoparticle compositions. 100 μL of the diluted formulation in 1×PBS isadded to 900 μL of a 4:1 (v/v) mixture of methanol and chloroform. Aftermixing, the absorbance spectrum of the solution is recorded, forexample, between 230 nm and 330 nm on a DU 800 spectrophotometer(Beckman Coulter, Beckman Coulter, Inc., Brea, Calif.). Theconcentration of therapeutic and/or prophylactic in the nanoparticlecomposition can be calculated based on the extinction coefficient of thetherapeutic and/or prophylactic used in the composition and on thedifference between the absorbance at a wavelength of, for example, 260nm and the baseline value at a wavelength of, for example, 330 nm.

For nanoparticle compositions including an RNA, a QUANT-IT™ RIBOGREEN®RNA assay (Invitrogen Corporation Carlsbad, Calif.) can be used toevaluate the encapsulation of an RNA by the nanoparticle composition.The samples are diluted to a concentration of approximately 5 μg/mL in aTE buffer solution (10 mM Tris-HCl, 1 mM EDTA, pH 7.5). 50 μL of thediluted samples are transferred to a polystyrene 96 well plate andeither 50 μL of TE buffer or 50 μL of a 2% Triton X-100 solution isadded to the wells. The plate is incubated at a temperature of 37° C.for 15 minutes. The RIBOGREEN® reagent is diluted 1:100 in TE buffer,and 100 μL of this solution is added to each well. The fluorescenceintensity can be measured using a fluorescence plate reader (WallacVictor 1420 Multilablel Counter; Perkin Elmer, Waltham, Mass.) at anexcitation wavelength of, for example, about 480 nm and an emissionwavelength of, for example, about 520 nm. The fluorescence values of thereagent blank are subtracted from that of each of the samples and thepercentage of free RNA is determined by dividing the fluorescenceintensity of the intact sample (without addition of Triton X-100) by thefluorescence value of the disrupted sample (caused by the addition ofTriton X-100).

C. In Vivo Formulation Studies

In order to monitor how effectively various nanoparticle compositionsdeliver therapeutic and/or prophylactics to targeted cells, differentnanoparticle compositions including a particular therapeutic and/orprophylactic (for example, a modified or naturally occurring RNA such asan mRNA) are prepared and administered to rodent populations. Mice areintravenously, intramuscularly, intraarterially, or intratumorallyadministered a single dose including a nanoparticle composition with aformulation such as those provided in Example 3. In some instances, micemay be made to inhale doses. Dose sizes may range from 0.001 mg/kg to 10mg/kg, where 10 mg/kg describes a dose including 10 mg of a therapeuticand/or prophylactic in a nanoparticle composition for each 1 kg of bodymass of the mouse. A control composition including PBS may also beemployed.

Upon administration of nanoparticle compositions to mice, dose deliveryprofiles, dose responses, and toxicity of particular formulations anddoses thereof can be measured by enzyme-linked immunosorbent assays(ELISA), bioluminescent imaging, or other methods. For nanoparticlecompositions including mRNA, time courses of protein expression can alsobe evaluated. Samples collected from the rodents for evaluation mayinclude blood, sera, and tissue (for example, muscle tissue from thesite of an intramuscular injection and internal tissue); samplecollection may involve sacrifice of the animals.

Nanoparticle compositions including mRNA are useful in the evaluation ofthe efficacy and usefulness of various formulations for the delivery oftherapeutic and/or prophylactics. Higher levels of protein expressioninduced by administration of a composition including an mRNA will beindicative of higher mRNA translation and/or nanoparticle compositionmRNA delivery efficiencies. As the non-RNA components are not thought toaffect translational machineries themselves, a higher level of proteinexpression is likely indicative of a higher efficiency of delivery ofthe therapeutic and/or prophylactic by a given nanoparticle compositionrelative to other nanoparticle compositions or the absence thereof.

Example 3: Sample Formulations

Nanoparticle compositions including a therapeutic and/or prophylacticcan be optimized according to the selection of a compound according toFormula (I), (IA), (II), (IIa), (IIb), (IIc), (IId) or (IIe), theselection of additional lipids, the amount of each lipid in the lipidcomponent, and the wt:wt ratio of the lipid component to the therapeuticand/or prophylactic, as described herein.

Initial studies were performed to compare the delivery efficiency ofnanoparticle compositions including various compounds according toFormula (I), (IA), (II), (IIa), (IIb), (IIc), (IId) or (IIe). Thecationic lipid MC3 is a current standard in the art. Accordingly, thestandard MC3 formulation including about 50 mol % MC3, about 10 mol %DSPC, about 38.5 mol % cholesterol, and about 1.5 mol % PEG-DMG was usedas a basis for this study. Nanoparticle compositions including DSPC as aphospholipid, cholesterol as a structural lipid, PEG-DMG as a PEG lipid,an RNA, and a compound according to Formula (I), (IA), (II), (IIa),(IIb), (IIc), (IId) or (IIe) selected from Compounds 1-159, 168-170, and173-175 were prepared according to or via methods similar to thosedescribed in Examples 1 and 2. The ratios of the lipids were50:10:38.5:1.5 mol % for the lipid according to Formula (I), (IA), (II),(IIa), (IIb), (IIc), (IId) or (IIe):DSPC:cholesterol:PEG-DMG. The RNAused was an mRNA encoding G5 luciferase (Luc) or G5 hEPO. Tables 1A-1Bsummarize the content and characteristics of the formulations.

As shown in Tables 1A-1B, the choice of compound according to Formula(I), (IA), (II), (IIa), (IIb), (IIc), (IId) or (IIe) dramaticallyaffects the size (e.g., diameter), polydispersity index, andencapsulation efficiency (EE) of the compositions. Compositions hadsizes between approximately 53 nm and 237 nm. Compositions includingCompounds 5, 35, 36, 51, 59, 131, 132, 137-139, 145, 148, 155 and 158produced the largest particles, while compositions including Compounds9, 21, 29, 30, 65, 7175, 94, 107, 114-116, 119, 124, 133, 149, 150, 152,174 and 175 produced the smallest particles. Polydispersity indicesvaried between 0.04 and 0.99, while encapsulation efficiencies exceeded75% for compositions including every tested compound except forCompounds 21, 94107, 132, 148, 155 and 158. The highest encapsulationefficiencies were observed for Compounds 1, 6, 18, 19, 24, 26, 28, 29,49, 50, 55, 60, 61, 65-70, 72, 74, 75, 101, 109-116, 118, 119, 121, 122,124, 126, 128, 130, 149, 152, 153, 156, 159, 169, 170 and 174.

TABLE 1A Characteristics of nanoparticle compositions includingcompounds according to Formula (I), (IA), (II), (IIa), (IIb), (IIc),(IId) or (IIe). Compound Size (nm) PDI EE (%) pKa  1 72.7 0.091 97.046.50  2 83.9 0.14 93.88 6.73  3 97.5 0.20 92.25 6.72  4 120.5 0.21 95.106.33  5 196.4 0.21 77.07 6.84  6 73.1 0.066 97.60 6.32  7 118.9 0.2286.10 6.75  8 121.0 0.15 95.8 6.64  9 68.5 0.12 75.7 4.87  10 102.9 0.1889.60 6.09  11 129.6 0.13 92.47 5.97  12 116.7 0.17 92.44 5.99  13 79.40.13 92.28 5.67  14 130.1 0.15 95.24 6.58  15 111.1 0.094 92.47 5.58  16119.0 0.16 91.32 5.52  17 85.2 0.24 91.84 7.76  18 86.2 0.042 97.50 6.56 19 101.1 0.17 97.21 6.78  20 111.5 0.13 96.72 6.87  21 53.5 n.d. −15.1n.d.  22 80.2 0.22 96.00 6.21  23 104.5 0.09 92.68 6.84  24 99.5 0.1397.16 6.71  25 85.8 0.10 95.80 6.68  26 91.9 0.16 97.43 6.64  27 82.30.18 94.27 6.78  28 99.4 0.20 97.03 6.04  29 66.8 0.11 96.99 6.00  3059.4 0.15 95.69 6.75  31 73.9 0.15 95.11 6.64  32 105.6 0.18 94.87 6.75 33 107.3 0.13 95.66 6.80  34 133.8 0.14 92.52 6.64  35 151.1 0.18 90.826.85  36 163.5 0.17 81.45 7.38  47 80.6 0.10 96.40 n.d.  48 82.3 0.09296.55 6.68  49 73.1 0.110 96.86 6.52  50 68.4 0.100 97.33 6.42  51 148.80.17 89.83 n.d.  52 130.5 0.19 93.25 n.d.  53 125.4 0.13 95.8 n.d.  54112.9 0.19 96.71 6.51  55 91.6 0.16 97.03 6.44  56 112.1 0.17 95.18 n.d. 57 128.4 0.16 94.33 n.d.  58 130.8 0.14 92.54 n.d.  59 237.0 0.24 94.44n.d.  60 95.1 0.12 97.6 6.73  61 89.1 0.11 97.2 6.70  65 63.9 0.12 98.26.36  66 76.7 0.120 96.52 76.7  67 77 0.13 98 6.38  68 76.8 0.14 97.76.69  69 77.2 0.13 98.4 6.92  70 73.7 0.15 97.5 6.51  71 60.1 0.11 96.15.88  72 65.4 0.11 97.3 6.29  73 59.2 0.13 95.7 5.95  74 65.6 0.15 976.08  75 64.2 0.10 98.1 6.67  79 93.7 0.18 89.1 7.53  80 118 0.19 90.77.52  81 99.2 0.14 95.4 7.14  94 62.4 0.24 0 4.43  96 120.5 0.160 79.046.600 101 91.7 0.230 98.96 7.27 103 78.8 0.160 90.77 6.13 107 55 0.74 04.802 108 119 0.14 96 7.17 109 81.1 0.13 98.6 6.78 110 118 0.13 97.48.03 111 79.3 0.14 98.2 7.13 112 85.7 0.12 99 7.78 113 69.2 0.15 99 6.93114 65.1 0.11 98.8 6.42 115 64.5 0.11 99.7 n.d. 116 63.3 0.14 99.4 5.66118 72.1 0.08 98 6.14 119 60.8 0.24 98.1 5.29 121 98.4 0.18 100 8.50 12269.3 0.09 98.2 6.83 123 81.6 0.23 94.4 6.27 124 61.3 0.1 97.7 5.89 12590.9 0.16 79.6 n.d. 126 77.4 0.18 96.8 6.00 127 110.4 0.19 89.5 6.98 12869.4 0.14 98.2 6.56 129 86.3 0.19 77.2 7.3 130 107.1 0.13 97 6.83 131167.9 0.095 75.44 7.76 132 298.0 0.180 30.77 7.34 133 66.0 0.098 91.486.38 134 85.6 0.110 94.62 6.66 135 89.5 0.130 90.20 6.47 136 140.4 0.590.9 6.95 137 184.4 <1 85.7 7.06 138 179.4 <0.5 91.8 7.39 139 174.0 0.5478.2 7.04 140 120.3 0.84 89.2 7.71 141 91.3 0.99 94.1 7.47 143 93.3 0.1996.4 6.47 144 135.9 0.22 90.3 7.09 145 176.5 0.140 89.15 7.25 146 97.00.210 91.94 7.78 147 99.5 0.130 88.31 6.66 148 192.7 0.200 25.49 6.646149 62.1 0.110 98.00 6.284 150 63.1 0.082 96.72 6.101 151 105.7 0.14087.86 6.593 152 62.6 0.072 99.29 6.465 153 83.7 0.150 98.39 6.580 15492.9 0.110 94.28 6.827 155 208.3 0.240 37.36 6.576 156 74.3 0.072 98.906.572 157 69.6 0.096 96.43 6.275 158 251.8 0.080 35.70 6.953 159 75.90.190 99.29 7.873 168 80.7 0.1 94.35 n.d. 169 75.4 0.18 99.04 n.d. 17071.7 0.12 98.24 n.d. 173 75.5 0.16 92.89 n.d. 174 61.4 0.12 98.52 n.d.175 65.4 0.2 93.23 n.d. MC3 79.7 0.11 97.3 n.d. n.d. = not determined

TABLE 1B Characteristics of nanoparticle compositions includingcompounds according to Formula (I), (IA), (II), (IIa), (IIb), (IIc),(IId) or (IIe). Endotoxin Apparent Compound Size (nm) PDI EE (%) (EU/mL)pKa  18^(#) 73.7 0.14 96.95 <1 6.56  25^(#) 69.7 0.14 97.92 1.8 6.68 30^(#) 76.3 0.13 96.32 <1 6.75 108^(#) 89.6 0.22 95.38 <1 7.17 109^(#)75 0.099 98.29 <1 6.78 110^(#) 73.3 0.24 92.39 <1 8.03 111^(#) 93.3 0.1391.23 1.4 7.13 112^(#) 60.6 0.21 96.40 1.8 7.78  60^(#) 88.9 0.15 95.20<1 6.73 122^(#) 70.2 0.12 96.27 1.2 6.83 MC3^(#) 57.7 0.12 99.01 <1 6.35^(#) = Formulated with hEPO mRNA

Example 4: Expression of Luc Induced by Sample Formulations

The efficacy of the nanoparticle compositions presented in Table 1A wasevaluated with a bioluminescence study. Formulations were administeredintravenously to mice (n=6) at a dosage of 0.5 mg/kg (mpk) andbioluminescence measured at 3, 6, and 24 hour time points. The standardMC3 formulation and, in some instances, a control (e.g., a PBS control)were evaluated for comparison. As is evident in Table 2, at 3 hours, thetotal flux was highest for compositions including Compounds 4, 28, 32,48, 66, 128 and 135 and the total flux at 3 h was higher than orcomparable to that of MC3 formulations for Compounds 2, 3, 18, 19, 20,24, 26, 25, 27, 31, 33, 47, 49, 50, 53-55, 60, 61, 65-68, 70, 72, 74,75, 96, 111, 122, 130, 133, 134, 143, 147, 148, 150, 151 and 153. Thesecompositions also demonstrated higher total flux at 6 and 24 hour timepoints. Compositions including Compounds 9, 17, 57, 58, 59, 121, 125,137, 140, 141 and 158 had significantly lower flux at all time pointsmeasured. In general, flux decreased as time progressed to less than 10%of the initial flux. These results suggest that the compounds describedherein may be useful in transfection applications.

TABLE 2 Expression of luciferase induced by administration ofnanoparticle compositions including compounds according to Formula (I),(IA), (II), (IIa), (IIb), (IIc), (IId) or (IIe). Total Flux Compound 3hours 6 hours 24 hours  1 3.48E+09 3.40E+09 4.10E+08  2 1.93E+104.31E+10 2.43E+09  3 6.55E+10 7.37E+10 4.96E+09  4 1.37E+11 6.01E+101.13E+09  5 2.77E+08 1.76E+08 2.40E+07  6 5.38E+09 7.60E+09 7.69E+08  74.13E+10 4.03E+10 1.68E+09  8 7.43E+09 6.71E+09 7.84E+08  9 1.43E+083.46E+06 1.01E+06  10 6.03E+08 2.37E+09 4.04E+07  11 3.38E+09 7.11E+091.15E+08  12 5.14E+09 1.27E+10 2.45E+08  13 1.02E+08 1.56E+08 1.47E+06 14 4.43E+08 2.29E+09 1.39E+08  15 4.31E+08 4.41E+07 2.05E+06  162.58E+08 5.45E+08 2.37E+07  17 7.72E+06 3.58E+06 6.79E+05  18 1.71E+102.13E+10 2.51E+09  19 3.38E+10 3.56E+09 4.68E+08  20 1.71E+10 2.48E+105.40E+08  22 6.57E+08 3.89E+08 2.73E+07  23 1.83E+09 1.15E+09 3.71E+08 24 1.72E+10 2.25E+10 1.83E+09  25 2.27E+10 1.59E+10 9.77E+08  266.75E+10 1.57E+10 1.54E+09  27 1.64E+10 1.03E+10 1.94E+09  28 8.98E+101.13E+11 1.20E+09  29 4.61E+09 2.89E+09 3.55E+08  30 1.19E+10 2.09E+101.21E+09  31 4.19E+10 5.31E+10 1.68E+09  32 8.65E+10 6.08E+10 1.92E+09 33 6.53E+10 1.20E+11 3.71E+09  34 1.06E+10 1.48E+10 6.69E+08  359.82E+08 1.24E+09 5.09E+07  36 6.97E+07 1.72E+08 4.44E+05  47 6.55E+105.38E+10 2.09E+09  48 8.73E+10 1.10E+11 2.92E+09  49 4.48E+10 1.08E+111.24E+09  50 3.81E+10 7.49E+10 5.02E+08  51 1.34E+08 2.80E+08 6.20E+06 52 2.91E+09 4.63E+09 2.55E+07  53 1.91E+10 2.32E+10 1.01E+09  545.36E+10 4.18E+10 9.07E+08  55 5.07E+10 1.68E+10 4.06E+08  56 1.27E+108.06E+09 2.53E+08  57 6.69E+06 6.21E+06 4.16E+05  58 5.69E+05 7.60E+053.64E+05  59 2.75E+05 2.79E+05 1.45E+05  60 7.91E+10 9.04E+10 2.90E+09 61 6.54E+10 6.20E+10 1.78E+09  65 6.56E+10 7.01E+10 7.50E+08  669.66E+10 4.577E+10  5.56E+09  67 4.24E+10 4.62E+10 4.51E+08  68 5.22E+108.16E+10 2.15E+09  69 3.38E+09 7.95E+09 1.15E+08  70 4.70E+10 2.49E+109.27E+08  71 4.09E+09 9.28E+09 6.51E+07  72 1.73E+10 4.07E+10 7.12E+08 73 8.10E+09 1.07E+10 1.27E+08  74 3.27E+10 2.23E+10 2.75E+08  753.51E+10 8.80E+10 2.13E+09  79 3.23E+08 5.27E+08 3.08E+07  80 2.76E+083.26E+08 1.54E+07  81 7.87E+09 9.96E+09 5.13E+08  96 4.54E+10 1.05E+113.86E+09 101 1.89E+08 1.41E+08 3.64E+06 103 2.68E+09 1.82E+09 9.45E+07108 5.04E+09 5.53E+09 1.50E+08 109 3.82E+09 4.88E+09 8.06E+07 1101.89E+09 2.57E+09 1.11E+08 111 1.89E+10 3.57E+10 8.86E+08 112 9.69E+081.04E+09 2.75E+07 113 5.16E+09 8.09E+09 1.30E+08 114 8.41E+07 5.98E+07n.d. 115 2.13E+07 2.91E+07 n.d. 116 3.13E+07 3.86E+07 n.d. 118 1.46E+091.16E+09 4.37E+07 119 1.02E+07 3.74E+07 n.d. 121 1.29E+06 1.36E+06 n.d.122 3.64E+10 8.64E+10 1.95E+09 123 4.06E+09 1.81E+10 5.18E+08 1246.62E+07 3.91E+09 5.13E+06 125 2.44E+05 3.16E+05 n.d. 126 7.59E+091.09E+10 1.40E+08 127 3.81E+09 2.09E+09 4.56E+08 128 1.04E+11 8.99E+101.00E+09 129 5.97E+09 4.51E+09 2.22E+08 130 6.26E+10 8.92E+10 1.08E+09131 6.97E+09 7.64E+09 2.47E+08 132 1.77E+09 1.36E+09 5.31E+07 1333.32E+10 2.93E+10 4.74E+08 134 2.01E+10 2.91E+10 8.00E+08 135 1.24E+119.90E+10 2.51E+09 136 7.21E+08 7.33E+08 3.39E+07 137 3.77E+05 5.02E+054.49E+05 138 2.97E+07 2.30E+07 1.63E+06 139 3.50E+07 1.17E+07 5.89E+05140 3.74E+06 1.70E+06 5.67E+05 141 2.16E+06 1.21E+06 3.49E+05 1431.76E+10 2.03E+10 2.47E+08 144 9.50E+09 1.82E+09 3.36E+08 145 7.11E+096.50E+09 2.38E+08 146 9.48E+07 8.39E+07 2.30E+06 147 3.24E+10 4.87E+103.32E+08 148 6.28E+10 3.71E+10 1.43E+09 149 1.01E+10 8.33E+09 3.45E+08150 1.66E+10 2.31E+10 3.86E+08 151 5.63E+10 5.68E+10 2.23E+09 1521.56E+09 2.45E+09 4.95E+07 153 1.69E+10 2.28E+10 5.10E+08 154 2.49E+094.89E+09 6.26E+07 155 2.49E+09 1.15E+10 1.99E+08 156 5.68E+09 1.03E+106.53E+07 157 8.54E+09 2.22E+10 1.90E+08 158 2.69E+05 9.82E+05 1.55E+05159 3.32E+06 1.20E+07 4.98E+05 MC3 1.58E+10 2.12E+10 7.19E+08 n.d. = notdetermined

The total flux (measured by area under the curve, AUC) induced byadministration of a formulation including a given lipid relative to thatinduced by administration of a formulation including MC3 was alsomeasured for several lipids. As shown in Table 3A (i.v. administration),the flux induced by formulations including Compounds 48 and 49 measuredat 6 h was ten times higher than that induced by the MC3 formulation.Formulations including Compounds 50, 54, and 55 also demonstrated higherflux than MC3 formulations. As shown in Table 3B, the flux induced byformulations including Compounds 108 and 168 measured at 6 h wasfourteen and sixteen times higher than that induced by the MC3formulation via intramuscular administration (i.m.). Results are alsoshown in FIG. 8. As shown in Table 3C (i.v. administration), the fluxinduced by formulations including Compounds 66, 133-135, and 147measured at 6 h and the total flux were noticeably higher than thoseinduced by the MC3 formulation. As shown in Table 3D, the total fluxinduced by formulations including Compounds 96, 148, and 151 measured at6 h was noticeably higher than that induced by the MC3 formulation.

TABLE 3A Expression of luciferase upon administration of formulationsincluding compounds according to Formula (I), (IA), (II), (IIa), (IIb),(IIc), (IId) or (IIe) relative to administration of formulationsincluding MC3. Compound Fold increase in total body Luc Flux relative toMC3 at 6 h 1 0.40 2 1.31 3 2.24 4 1.31 5 0.005 6 1.15 16 0.02 18 3.22 190.96 20 0.80 24 2.67 25 1.89 26 4.24 27 0.31 28 2.46 29 0.78 30 2.49 311.21 32 1.39 33 2.74 34 0.34 35 0.028 36 0.004 48 10.0 49 9.81 50 6.8151 0.025 53 2.11 54 3.80 55 1.52 56 0.733 57 0.00056 58 0.00007 590.00003 65 3.16 66 0.103 67 2.08 68 3.68 71 0.418 73 0.48 74 1.005 1270.094 128 4.05 129 0.203 130 4.02 MC3 1.00

TABLE 3B Expression of luciferase upon administration of formulationsincluding compounds according to Formula (I), (IA), (II), (IIa), (IIb),(IIc), (IId) or (IIe) relative to administration of formulationsincluding MC3. i.v. i.m. Lipid/MC3 Lipid/MC3 Compound 0.5 mpk, Luc, 6 h0.01 mpk, Luc, 6 h 108 0.4 14.2 109 0.3 3.6 111 2.6 4.9 168 ND 16.0 169ND 3.3 170 ND 5.3 173 ND 0.6 174 ND 0.2 175 ND 0.1 ND = not determined

TABLE 3C Expression of luciferase upon administration of formulationsincluding compounds according to Formula (I), (IA), (II), (IIa), (IIb),(IIc), (IId) or (IIe) relative to administration of formulationsincluding MC3. Fold 6 h Lipid/MC3 6 h Lipid/MC3 increase Avg. Luc G.M.Luc AUC AUC Compound Expression Expression (h * p/s) Lipid/MC3  66 6.285.23 6.76E+11 8.20 101 0.019 0.011  1.8E+09 0.022 103 0.250 0.171 2.4E+10 0.291 131 1.05 1.27 9.29E+10 1.13 132 0.187 0.112 1.75E+100.212 133 4.02 4.18 3.62E+11 4.39 134 3.99 3.27 3.43E+11 4.16 135 13.610.5 1.25E+12 15.2 145 0.89 0.979  8.1E+10 0.983 146 0.011 0.0121.04E+09 0.013 147 6.68 8.13 5.63E+11 6.83 MC3 1 1 8.24E+10 1 Avg. =average; G.M. = geometric mean

TABLE 3D Expression of luciferase and lipid clearance uponadministration of formulations including compounds according to Formula(I), (IA), (II), (IIa), (IIb), (IIc), (IId) or (IIe) relative toadministration of formulations including MC3. % Dose % Dose RemainingRemaining Lipid/MC3 in Liver in Liver Lipid AUC (p/s * h) AUC 6 h 6 h148 4.965E+011 2.6 <1 <1 149 1.057E+011 0.55 150 2.704E+011 1.4 <1 <1 96 1.209E+012 6.3 <1 <1 151 7.010E+011 3.7 <1 <1 152 2.855E+010 0.15153 2.697E+011 1.4 <1 <1 154 5.560E+010 0.29 155 1.266E+011 0.66 1561.170E+011 0.61 157 2.481E+011 1.3 <1 <1 158 1.211E+007 <0.01 1591.355E+008 <0.01 MC3 1.909E+011 1 80 54

Example 5: Expression of Luc Induced by Sample Formulations in DifferentOrgans

The efficacy of the nanoparticle compositions presented in Table 1A wasfurther evaluated by measuring the expression of modified luciferase inthe liver, lung, spleen, and femur upon administration of a givencomposition. Formulations were administered intravenously to mice (n=3)at a dosage of 0.5 mpk and bioluminescence measured after 6 hours. Thestandard MC3 formulation and a PBS control were also tested. As isevident in Table 4, flux for nearly all species was higher in the livercompared to other tissues. Flux in the liver was highest forcompositions including 3, 28, 33, 48, 96 and 135 and comparable to thatof MC3 formulations for compositions including Compounds 2, 4, 6, 7, 18,20, 24-27, 30-32, 34, 47, 49, 50, 53-56, 60, 65, 67, 68, 74, 75, 111,113, 122, 128, 130, 133, 134, 143, 147-151, 153 and 157. Flux in theliver was lowest for compositions including Compounds 58, 59, 137, and141. Flux in the spleen was highest for compositions including Compounds4, 7, 33, 34, 48, 53, 108, 129, 130, and 148, and lowest forcompositions including Compounds 9, 59, 124, and 141. Similar resultswere observed in the lung and femur.

TABLE 4 Expression of luciferase in various organs 6 hours afteradministration of nanoparticle compositions including compoundsaccording to Formula (I), (IA), (II), (IIa), (IIb), (IIc), (IId) or(IIe). Total Flux Compound Liver Lung Spleen Femur 1 4.02E+08 1.72E+064.27E+06 7.52E+05 2 4.87E+09 2.52E+07 5.77E+07 3.86E+06 3 1.39E+104.76E+07 1.47E+08 7.36E+06 4 5.26E+09 6.22E+07 4.09E+08 n.d. 5 5.84E+071.89E+06 1.55E+08 1.22E+06 6 1.09E+09 4.30E+06 3.03E+07 2.15E+06 72.49E+09 3.95E+07 4.83E+08 n.d. 8 7.87E+08 4.06E+06 1.51E+08 n.d. 94.30E+05 2.56E+04 5.51E+04 2.57E+04 10 3.22E+08 8.85E+05 8.17E+065.09E+05 11 8.03E+08 1.35E+07 1.04E+08 n.d. 12 6.84E+08 7.45E+066.82E+07 n.d. 13 2.25E+07 2.21E+05 7.09E+05 1.35E+05 14 1.91E+084.74E+06 1.92E+08 4.91E+06 15 6.23E+06 6.41E+04 9.01E+05 5.93E+04 163.17E+07 4.18E+05 5.43E+06 2.55E+05 17 5.52E+05 9.95E+04 5.58E+069.55E+04 18 2.76E+09 1.25E+07 5.15E+07 4.68E+06 19 6.33E+08 5.99E+061.77E+07 1.68E+06 20 1.84E+09 2.66E+07 1.43E+08 1.31E+07 22 4.00E+074.73E+05 1.57E+06 1.16E+05 23 2.92E+08 1.82E+06 3.08E+07 1.19E+06 244.19E+09 1.71E+07 8.78E+07 4.54E+06 25 2.41E+09 1.51E+07 3.11E+074.40E+06 26 2.90E+09 1.18E+07 1.56E+07 4.67E+06 27 2.16E+09 6.35E+063.78E+06 2.00E+06 28 1.22E+10 2.17E+08 1.80E+08 n.d. 29 5.20E+089.83E+05 5.99E+06 9.56E+05 30 2.68E+09 1.02E+07 3.55E+07 6.38E+06 315.17E+09 7.55E+06 9.42E+07 n.d. 32 8.52E+09 1.16E+07 1.70E+08 n.d. 331.78E+10 2.92E+07 3.77E+08 n.d. 34 2.08E+09 9.49E+06 2.40E+08 n.d. 351.63E+08 2.06E+06 1.23E+08 n.d. 36 2.65E+07 5.82E+05 6.14E+07 n.d. 474.86E+09 8.71E+06 8.33E+07 n.d. 48 1.08E+10 3.31E+07 3.49E+08 n.d. 495.68E+09 2.52E+07 1.87E+08 n.d. 50 6.30E+09 2.81E+07 1.14E+08 n.d. 512.49E+07 3.67E+05 2.80E+07 n.d. 52 5.86E+08 2.80E+06 8.30E+07 n.d. 532.02E+09 2.47E+07 8.54E+08 n.d. 54 5.57E+09 1.12E+07 1.64E+08 n.d. 551.92E+09 7.02E+06 2.63E+07 n.d. 56 1.04E+09 4.62E+06 1.98E+08 n.d. 579.36E+05 3.18E+04 2.47E+06 n.d. 58 8.71E+04 1.21E+04 2.38E+05 n.d. 592.87E+05 4.41E+04 9.68E+04 n.d. 60 1.54E+09 6.25E+06 7.12E+06 n.d. 616.37E+08 3.56E+06 1.61E+07 n.d. 65 9.56E+09 3.79E+07 6.57E+07 n.d. 665.01E+09 4.20E+06 2.00E+07 n.d. 67 3.60E+09 1.68E+07 2.55E+07 n.d. 688.42E+09 3.98E+07 6.69E+07 n.d. 69 2.24E+08 7.34E+05 2.54E+06 n.d. 708.55E+08 6.32E+06 2.06E+06 n.d. 71 7.93E+08 4.86E+06 8.04E+06 n.d. 727.97E+08 1.05E+07 6.40E+06 n.d. 73 7.93E+08 6.17E+06 9.45E+06 n.d. 741.99E+09 6.93E+06 2.26E+07 n.d. 75 1.45E+09 3.92E+06 5.66E+06 n.d. 793.15E+06 6.13E+04 6.45E+05 n.d. 80 1.09E+07 8.97E+04 4.71E+06 n.d. 812.74E+08 6.23E+06 4.49E+07 n.d. 96 1.56E+10 3.43E+07 3.39E+08 n.d. 1011.27E+07 1.77E+05 5.60E+06 n.d. 103 8.48E+07 2.06E+05 2.65E+06 n.d. 1084.63E+08 9.81E+06 7.82E+08 n.d. 109 8.17E+08 6.03E+06 4.81E+07 n.d. 1102.30E+08 5.76E+06 1.41E+08 n.d. 111 4.83E+09 2.57E+07 2.44E+08 n.d. 1121.48E+08 1.83E+06 2.75E+07 n.d. 113 1.11E+09 5.55E+06 5.22E+07 n.d. 1181.72E+08 1.98E+06 2.49E+07 n.d. 122 2.63E+09 2.77E+07 1.56E+07 n.d. 1232.50E+08 1.78E+06 4.04E+06 n.d. 124 8.46E+06 5.67E+04 8.06E+04 n.d. 1267.41E+08 2.68E+06 1.87E+07 n.d. 127 1.94E+08 5.26E+06 3.21E+08 n.d. 1285.98E+09 2.16E+07 7.09E+07 n.d. 129 6.65E+08 9.89E+06 5.09E+08 n.d. 1308.17E+09 5.88E+07 1.35E+09 n.d. 131 3.52E+08 1.45E+07 8.32E+08 n.d. 1321.49E+08 1.39E+07 3.37E+08 n.d. 133 2.94E+09 3.18E+06 1.77E+07 n.d. 1341.73E+09 2.82E+06 1.85E+07 n.d. 135 1.65E+10 2.71E+07 1.39E+08 n.d. 1361.34E+08 8.91E+05 2.77E+07 6.60E+05 137 6.48E+04 1.66E+04 1.32E+052.02E+04 138 3.66E+06 9.47E+04 4.04E+06 1.58E+05 139 8.27E+05 5.26E+042.10E+06 5.12E+04 140 4.21E+05 2.14E+04 2.22E+05 3.26E+04 141 1.59E+053.85E+04 6.29E+04 2.86E+04 143 1.76E+09 3.60E+07 1.42E+08 n.d. 1443.75E+08 4.81E+06 5.11E+07 2.44E+06 145 5.01E+08 1.36E+07 4.25E+08 n.d.146 7.24E+06 3.88E+06 5.11E+07 n.d. 147 5.24E+09 6.73E+06 8.57E+07 n.d.148 4.39E+09 3.27E+07 2.71E+09 n.d. 149 1.11E+09 2.69E+06 2.71E+07 n.d.150 1.54E+09 2.20E+06 3.43E+07 n.d. 151 4.72E+09 9.20E+06 9.27E+07 n.d.152 1.43E+08 3.16E+05 6.63E+06 n.d. 153 1.18E+09 6.42E+06 1.42E+08 n.d.154 3.62E+08 2.89E+06 1.30E+07 n.d. 155 8.58E+08 1.00E+07 2.77E+08 n.d.156 6.51E+08 1.92E+06 1.82E+07 n.d. 157 2.27E+09 6.70E+06 5.15E+07 n.d.158 1.99E+05 1.71E+04 1.17E+05 n.d. 159 1.13E+06 2.17E+05 7.24E+05 n.d.MC3 2.57E+09 1.27E+07 2.85E+07 2.56E+06 n.d. = not determined

Example 6A: Expression Induced by Sample Formulations Upon IntramuscularAdministration

Sample formulations including both modified luciferase (Luc) mRNA andH10 mRNA were prepared and administered intramuscularly and theresulting expression and immunogenicity were evaluated simultaneously.Formulations including compounds according to Formula (I), (IA), (II),(IIa), (IIb), (IIc), (IId) or (IIe) were prepared and administered atdoses of 0.001 and 0.01 mpk (e.g., doses of 0.0005 mpk of a formulationincluding Luc mRNA and a formulation including H10 mRNA or doses of0.005 mpk of a formulation including Luc mRNA and a formulationincluding H10 mRNA). As shown in Table 5A, Compound 20 exhibited thehighest expression at both dose levels. The low dose of Compound 20showed equivalent expression to the high dose of MC3. Formulationsincluding other compounds also showed multi-fold enhancement inexpression relative to MC3.

TABLE 5A Total flux (p/s) measured 6 hours after intramuscularadministration of nanoparticle compositions including compoundsaccording to Formula (I), (IA), (II), (IIa), (IIb), (IIc), (IId) or(IIe). Compound 0.001 mpk Dose 0.01 mpk Dose 2 3.55E+06 6.16E+07 33.58E+06 4.95E+07 5 9.84E+05 3.55E+06 7 3.65E+06 7.48E+07 8 7.81E+053.32E+06 12 8.02E+04 8.90E+05 18 n.d. 8.84E+07 19 3.28E+06 2.96E+07 202.59E+07 9.72E+07 23 8.27E+06 2.20E+06 24 3.78E+06 3.97E+07 25 3.53E+069.96E+07 26 3.90E+06 6.13E+07 27 2.55E+06 3.17E+07 28 6.73E+05 5.56E+0629 7.64E+05 1.12E+07 30 2.47E+06 3.77E+07 32 7.37E+05 1.03E+07 352.45E+06 8.12E+06 48 4.69E+05 8.78E+06 50 6.56E+05 1.13E+07 57 1.16E+052.23E+05 137 7.57E+04 8.09E+04 138 2.72E+05 1.19E+06 140 2.03E+056.09E+05 144 2.72E+06 2.18E+07 MC3 2.76E+06 3.68E+07 n.d. = notdetermined

Example 6B: Expression Induced by Sample Formulations Upon IntramuscularAdministration

Sample formulations including modified luciferase (Luc) mRNA preparedand administered intramuscularly and the resulting expression andimmunogenicity were evaluated simultaneously. Formulations includingcompounds according to Formula (I), (IA), (II), (IIa), (IIb), (IIc),(IId) or (IIe) were prepared and administered at dose of and 0.01 mpk.As shown in Table 5B, Compound 108 exhibited the highest expression.Formulations including other compounds also showed multi-foldenhancement in expression relative to MC3.

TABLE 5B Total flux (p/s) measured 6 hours after intramuscularadministration of nanoparticle compositions including compoundsaccording to Formula (I), (IA), (II), (IIa), (IIb), (IIc), (IId) or(IIe). Compound 0.01 mpk Dose  60 9.48E+07  69 8.83E+06 108 4.60E+08 1091.18E+08 110 1.21E+08 111 1.58E+08 112 9.47E+07 114 3.31E+06 1211.06E+06 122 9.19E+07 123 1.08E+07 MC3 3.23E+07

The fluxes measured upon intravenous and intramuscular administrationare compared in Table 6. Fluxes are presented as fold increase over thatmeasured for MC3 formulations. Formulations including Compound 20displayed the highest fold increase in Luc expression upon intramuscularadministration, while those including Compounds 18 and 26 displayed thehighest fold increase upon intravenous administration. Notably, theintravenous data included in Table 6 was measured at higher doses thanthe intramuscular data.

TABLE 6 Relative flux measured after intravenous or intramuscularadministration of nanoparticle compositions including compoundsaccording to Formula (I), (IA), (II), (IIa), (IIb), (IIc), (IId) or(IIe). Fold increase in Luc expression relative to MC3 IntramuscularIntravenous Intramuscular (0.001 mpk Compound pKa (0.5 mpk dose) (0.01mpk dose) dose) 3 6.72 2.24 1.13 0.51 18 6.56 3.23 2.01 n.d. 20 6.870.80 2.21 3.70 26 6.64 4.24 1.39 0.56 29 6.00 1.03 0.25 0.11 n.d. = notdetermined

Example 7: Cytokine Production Induced by Sample Formulations

The introduction of foreign material into a mammalian body induces aninnate immune response that promotes cytokine production. Such immuneresponses to, for example, nanoparticle compositions includingtherapeutic and/or prophylactics, are undesirable. The induction ofcertain cytokines is thus measured to evaluate the efficacy ofnanoparticle compositions. The concentrations of various cytokines inmice upon intravenous administration of nanoparticle compositionspresented in Table 1A at a dosage of 0.5 mpk was measured at 6 hours.The standard MC3 formulation and a PBS control were also tested. As isevident in Table 7, IL-6 induction was highest for compositionsincluding Compounds 1, 3, 9, 19, and 26, while IP-10 induction washighest for compositions including Compounds 3, 4, 7, 20, and 26. IL-6induction was lowest for compositions including Compounds 4, 11, 12, and28. IP-10 induction was lowest for compositions including Compounds 10,11, 12, 13, 15, 17, and 18.

TABLE 7 Cytokine induction 6 hours after administration of nanoparticlecompositions including compounds according to Formula (I), (IA), (II),(IIa), (IIb), (IIc), (IId) or (IIe). Compound IL-6 IP-10 1 267.24 687.142 70.95 468.86 3 282.88 2052.87 4 13.1375 2253.09 5 94.07 487.16 6136.18 316.01 7 116.35 4959.16 9 317.45 366.53 10 88.81 138.16 11 0.1444.84 12 3.88 32.03 13 29.07 126.29 14 75.29 621.49 15 64.65 184.30 1632.01 206.75 17 138.43 156.41 18 78.76 139.92 19 285.56 1468.94 20126.83 2468.24 22 90.54 976.50 23 94.00 1015.95 24 163.53 1172.93 25233.45 1194.13 26 273.56 2330.01 27 161.07 345.56 28 17.47 283.13 2969.54 1362.81 30 152.51 1638.77 136 28.69 887.91 137 130.82 234.35 13823.38 172.56 139 23.57 153.36 140 282.82 187.83 141 327.15 1072.04 1436.245 209.63 144 319.46 4220.55 MC3 124.42 504.90

Example 8: Complement Activation Induced by Sample Formulations

Complement activation assists in the clearance of pathogens from anorganism. As it is undesirable that a subject's body recognize ananoparticle composition as a foreign invader, low complement systemactivation upon administration of such a composition is preferred. Thecomplex sC5b-9 is a marker for the activation of the complement system.Thus, human cells were contacted in vitro with nanoparticle compositionsaccording to Table 1A and were evaluated for sC5b-9 levels. Table 8shows the fold increase in sC5b-9 levels relative to saline fornanoparticle compositions including Compounds 1, 6, 9, 18, 24, 25, 29,and 30. Compositions including Compounds 6 and 18 somewhat increasesC5b-9 levels relative to saline, while compositions including Compounds1, 9, 24, 29, and 30 slightly decrease sC5b-9 levels relative to saline.

TABLE 8 Fold increases in sC5b-9 levels upon administration ofnanoparticle compositions including compounds according to Formula (I),(IA), (II), (IIa), (IIb), (IIc), (IId) or (IIe). Fold increase Compoundversus saline  1 0.82  6 1.39  9 0.92 18 1.28 24 0.81 25 1.02 29 0.93 300.94 136  0.69 139  0.73 140  0.75 141  1.81 MC3 0.73

Example 9: Clinical Chemistry and Hematology

Sample formulations of nanoparticle compositions including differentlipids were administered intravenously to rat at a dose of 2 mpk. Theexpression of various clinical markers was evaluated at 48 h post doseand compared to that induced by administration of MC3 formulations orphosphate buffered saline (PBS).

TABLE 9 Levels of clinical markers induced by administration ofnanoparticle compositions including a compound of Formula (I), (IA),(II), (IIa), (IIb), (IIc), (IId) or (IIe). Concentration AlanineAspartate Compound aminotransferase aminotransferase NeutrophilsLymphocytes Monocytes 3 53.5 87.5 3388.5 12051 2103 24 51.5 90 1790.514100 1834 25 52 124.5 1998 15924 2122 30 56 95 3195 10408.5 877 MC3 339325 4962.5 19976 1429 PBS 55.5 108 920 8004 276

Example 10: Expression of hEPO Induced by Sample Formulations

Sample formulations of nanoparticle compositions including differentlipids are generally first evaluated according to Luc expression invivo. The activity of several such compositions was further evaluatedusing an mRNA encoding hEPO. Nanoparticle compositions includingCompounds 6, 18, 25, 30, 108-112, 60, and 122, or MC3 were preparedaccording to Example 2. As shown in Tables 10 and 1B supra, eachcomposition had a similar particle size and encapsulation efficiency.

TABLE 10 Characteristics of nanoparticle compositions includingcompounds according to Formula (I), (IA), (II), (IIa), (IIb), (IIc),(IId) or (IIe). Size EE Compound Formulation (nm) PDI (%)  6 Compound6:DSPC:Chol:PEG-DMG 70.5 0.082 97.84 (50:10:38.5:1.5) 18 Compound18:DSPC:Chol:PEG- 78.6 0.095 97.34 DMG (50:10:38.5:1.5) MC3MC3:DSPC:Chol:PEG-DMG 73.7 0.114 97.22 (50:10:38.5:1.5)

The expression of hEPO and cytokine induction in mice intravenouslyadministered a nanoparticle composition at a dose of 0.5 mpk weremeasured at 3, 6, and 24 hours. The resultant hEPO and cytokine levelsare summarized in Table 11A. Compositions including Compounds 6 and 18yielded higher hEPO concentrations than MC3 formulations at each timepoint. The expression of hEPO in mice intramuscularly administered ananoparticle composition from Table 1B at a dose of 0.01 mpk weremeasured at 3, 6, and 24 hours. The resultant hEPO levels are summarizedin Table 11B. Compositions including Compounds 18, 25, 30, 108-112, 60,and 122 yielded higher hEPO concentrations than MC3 formulations at 6 hrtime point. (see also FIG. 9.)

TABLE 11A Evaluation of nanoparticle compositions including compoundsaccording to Formula (I), (IA), (II), (IIa), (IIb), (IIc), (IId) or(IIe). Cytokine expression (pg/ml) hEPO expression (pg/ml) IL-6 Compound3 h 6 h 24 h IP-10 (6 h) (6 h)  6 2.31E+06 3.17E+06 1.11E+06 116.6610.15 18 3.00E+06 3.38E+06 1.80E+06 299.93 10.16 MC3 1.57E+06 1.83E+060.81E+06 117.94 19.85

TABLE 11B Evaluation of nanoparticle compositions including compoundsaccording to Formula (I), (IA), (II), (IIa), (IIb), (IIc), (IId) or(IIe). Fold increase in hEPO Compound concentration relative to MC3  188.6  25 7.1  30 9.2 108 3.7 109 5.3 110 1.2 111 10.6 112 1.6  60 11.2122 10.7 MC3 1

Table 12 compares the nanoparticle compositions including compoundsaccording to Formula (I), (IA), (II), (IIa), (IIb), (IIc), (IId) or(IIe) to the compositions including MC3 on the basis of expression andflux levels. As is evident in Table 12, both Compounds 6 and 18outperform MC3 in both hEPO expression and average total flux. Thus,these lipids may be useful in nanoparticle composition therapeutics.

TABLE 12 Comparison of nanoparticle compositions including compoundsaccording to Formula (I), (IA), (II), (IIa), (IIb), (IIc), (IId) or(IIe). Compound Compound 6 18 MC3 Average hEPO concentration 3.17 × 10⁶3.38 × 10⁶  1.83 × 10⁶ (pg/ml, 6 h) Fold increase in hEPO 1.73 1.85 1concentration relative to MC3 Average total flux (6 h, ffluc) 7.60 × 10⁹2.13 × 10¹⁰ 6.59 × 10⁹ Fold increase in average total 1.15 3.23 1 fluxrelative to MC3

Example 11: Expression of hEPO Induced by Sample Formulations in Rat andResidual Lipid Levels in the Liver

The expression of hEPO and cytokine induction in rats intravenouslyadministered a nanoparticle composition at a dose of 2.0 mpk wasmeasured at 6 h.

At 48 h liver tissue was harvested for lipid quantification. Topre-weighed tissues, Milli-Q water was added (900 μL water per 100 mgtissue). Tissues were homogenized using an Omni probe homogenizer untiluniform. 50 μL of samples and matrix calibration standards werealiquoted into a 96-well plate. 50 μL of blank matrix for matrix blanksand control blanks were aliquoted. 400 μL IS spiking solution weremanually added to all samples except matrix blanks. 400 μL of 50:50ACN:IPA were manually added to matrix blanks. The plate was covered andthe samples vortexed and centrifuged for 5 minutes at >3000 rpm. 200 μLof the samples were transferred into a clean 96-well plate for analysis.Samples were analyzed on a Waters Acquity UPLC using a HigginsAnalytical Clipeus C8 column (5 μM, 30×2.1 mm) and a gradient of either70-95% or 60-95% (Mobile Phase A: 5 mM ammonium formate in 50:50:1H₂O:MeOH:formic acid; Mobile Phase B: 5 mM ammonium formate in 100:1MeOH:formic acid) over 1.3 min at 1.2 mL/min (column temperature 55°C.). Detection was based on electrospray ionization (ESI) in positivemode using a Sciex API5500 Mass Spectrometer.

TABLE 13 Expression of hEPO induced by administration of nanoparticlecompositions including compounds according to Formula (I), (IA), (II),(IIa), (IIb), (IIc), (IId) or (IIe) in rat, 6 h, 2 mpk. Compound hEPOexpression (pg/mL)  3 1.74E+07 18 9.96E+06 24 1.44E+07 25 3.05E+07 301.63E+07 MC3 1.33E+07

TABLE 14 Cytokine induction 6 hours after administration of hEPOnanoparticle compositions including compounds according to Formula (I),(IA), (II), (IIa), (IIb), (IIc), (IId) or (IIe). Compound IP-10 (pg/mL) 3 542 18 517.3 24 323.5 25 533.5 30 214.5 MC3 688.3

TABLE 15 Liver levels in rats administered compositions includingcompounds according to Formula (I), (IA), (II), (IIa), (IIb), (IIc),(IId) or (IIe) after 48 h. Compound % remaining dose in liver, 48 h  314.2 18 <1 24 <1 25 1.3 30 <1 MC3 74

The expression of hEPO in rats intravenously administered a nanoparticlecomposition at a dose of 0.2 mpk or 2.0 mpk was measured at 6 hours.Table 16 summarize the ratio of hEPO expression levels using variousnanoparticle compositions as compared to the hEPO expression level usingMC3 formulation and the lipid levels in the liver measured 48 hoursafter administration, as described above. Tables 17 and 18 summarize thelipid levels in the liver and spleen measured 48 hours afteradministration of Compounds 28, 33, 53, and 54. Liver and spleen levelsrepresent the average values calculated for 3 rats in each group. As isshown in Tables 17 and 18, less than 10% of Compounds 28, 33, 53, and 54remained in the liver after 48 hours, while greater than 60% of MC3remained.

TABLE 16 Ratio of expression of hEPO and lipid levels remaining in liverafter 48 h. Lipid/MC3 % Lipid Remaining hEPO conc. ratio in liver, 48 h*Compound 0.2 mpk 2 mpk 2 mpk MC3 1 1 87 18 n.d. 0.81 0.018 25 2.41 2.131.32 24 1.75 1.01 0.016 30 1.87 1.14 <0.01  3 2.41 1.21 14 26 n.d. 4.9520 48 5.39 3.84 7.22 49 4.13 3.28 12.6 50 3.41 3.03 15.9 *Assuming 300 grat and 15 g liver

TABLE 17 Liver levels in rats administered 0.2 mpk doses of compositionsincluding compounds according to Formula (I), (IA), (II), (IIa), (IIb),(IIc), (IId) or (IIe) after 48 h. Liver level Spleen level Compound(ng/g) (ng/g) 28 49.6 268 33 n.d. 115 53 4810 1181 54 6067 6357 MC325033 9440

TABLE 18 Liver levels in rats administered 2 mpk doses of compositionsincluding compounds according to Formula (I) (IA), (II), (IIa), (IIb),(IIc), (IId) or (IIe) after 48 h. Liver level Spleen level Compound(ng/g) (ng/g) 28 665 551 33 103 287 53 47033 201333 54 56100 49367 MC3285333 129000Table 19 summarizes the hEPO expression, IP-10 induction, liver andspleen levels, and alanine aminotransferase (ALT) and aspartateaminotransferase (AST) measured upon intravenous administration offormulations including Compounds 48, 49, and 50 to rat at 0.2 and 2 mpkand hEPO mRNA. hEPO concentrations were measured 6 hours afteradministration, while cytokine induction and liver and spleen levelswere measured 48 hours after administration. hEPO and IP-10concentrations are presented in pg/ml, while liver levels are providedin ng/g. ALT and AST levels are presented in international units.

TABLE 19 hEPO expression, IP-10 induction, and liver levels measuredafter administration of compositions including compounds according toone of formulas (I), (IA), (II), (IIa), (IIb), (IIc), (IId) or (IIe).Compound 48 Compound 49 Compound 50 MC3 0.2 mpk 2 mpk 0.2 mpk 2 mpk 0.2mpk 2 mpk 0.2 mpk 2 mpk hEPO 4.06E+06 3.57E+07 3.17E+06 3.04E+072.62E+06 2.81E+07 7.68E+06 9.29E+06 expression (pg/ml) IP-10 134 970 66932 20 1065 2 596 induction (pg/ml) Liver 5448 34520 6490 61400 582279200 11300 140520 level (ng/g) Spleen 0.31 0.21 0.36 0.37 0.22 0.170.74 0.65 level (ng/g) ALT 59.6 66.0 54.0 77.8 59.2 78.8 63.6 79.6 AST140.8 131.2 99.4 132.4 143.2 158.4 134.8 139.0

Example 12: Dose Response of Sample Formulations in Rats

The expression of hEPO induced by intravenous administration to rats ofnanoparticle compositions at various doses was measured at 2, 4, 6, 8,24, and 48 hour time points. FIGS. 3-6 respectively summarize the hEPOexpression measured upon intravenous administration of formulationsincluding Compounds 26, 18, 25, and MC3 to rat at various doses. Thelipid levels of Compound 26 in the liver after 48 hours were about 19%.

FIG. 7 shows the area under the curve for compositions includingCompounds 18, 25, and 26 and MC3 at different dosages: 0.005 mpk, 0.01mpk, 0.025 mpk, 0.05 mpk, 0.1 mpk, 0.25 mpk, 0.5 mpk, 1 mpk or 2 mpk.

Example 13: Pharmacokinetics of Sample Formulations in Rats

The expression of hEPO and lipid levels in the liver and spleen in ratsintravenously administered a nanoparticle composition at a dose of 0.2mpk was measured at various timepoints. Compounds 18 and 25 wereselected for comparison with MC3. Lipids were formulated according tothe standard MC3 formulation described above. Rats were administeredintravenously a single 0.2 mpk dose and expression monitored at 0.25,0.5, 1, 2, 4, 8, 24, and 48 hours after administration.

TABLE 20 Expression of hEPO induced by administration of nanoparticlecompositions in rat, 6 h, 0.2 mpk. hEPO expression Compound Compound(pg/mL) 18 25 MC3 0.25 h   20227 0 0 0.5 h   20743 19553 42457 1 h194353 434299 93720 2 h 238107 2042807 524093 4 h 514807 3176560 6013078 h 915320 2631633 1536833 24 h  412051 869374 703619 48 h  52361 10308964687

TABLE 21 Lipid level in liver induced by administration of nanoparticlecompositions in rat, 6 h, 0.2 mpk. Compound Compound Lipid level (ng/g)18 25 MC3 0.25 h   5374 12037 13180 0.5 h   6023 16447 20500 1 h 605317900 16777 2 h 2037 11733 25967 4 h 839 6687 24730 8 h 296 2357 3263324 h  5 199 33000 48 h  5374 12037 13180

TABLE 22 Lipid level in spleen induced by administration of nanoparticlecompositions in rat, 6 h, 0.2 mpk. Compound Compound Lipid level (ng/g)18 25 MC3 0.25 h   1230 4037 4100 0.5 h   2017 6880 6237 1 h 3213 85904197 2 h 3070 13733 8613 4 h 3770 20400 11920 8 h 1345 10787 21200 24 h 271 2023 19067 48 h  92 1547 11563

Example 14: Optimization of Lipid:Therapeutic Agent Ratios

The relative amounts of lipid component and therapeutic and/orprophylactic in a nanoparticle composition can be optimized according toconsiderations of efficacy and tolerability. For compositions includingan RNA as a therapeutic and/or prophylactic, the N:P ratio can serve asa useful metric.

As the N:P ratio of a nanoparticle composition controls both expressionand tolerability, nanoparticle compositions with low N:P ratios andstrong expression are desirable. N:P ratios vary according to the ratioof lipids to RNA in a nanoparticle composition. Thus, the wt/wt ratio oftotal lipid to RNA is varied between 10:1, 15:1, 20:1, 32:1, 40:1, 50:1,and 60:1 for a lipid formulation including about 50 mol % of a compoundaccording to Formula (I), (IA), (II), (IIa), (IIb), (IIc), (IId) or(IIe), about 10 mol % phospholipid (e.g., DOPE or DSPC), about 38.5 mol% structural lipid (e.g., cholesterol), and about 1.5 mol % PEG lipid(e.g., PEG-DMG). N:P ratios are calculated for each nanoparticlecomposition assuming a single protonated nitrogen atom. Theencapsulation efficiency (EE), size, and polydispersity index of eachcomposition are also measured.

Generally, compositions with higher total lipid:RNA ratios yield smallerparticles with higher encapsulation efficiencies, both of which aredesirable. However, the N:P ratio for such formulations generallyexceeds 4. Current standards in the art such as the MC3 formulationdescribed above have N:P ratios of 5.67. Thus, a balance between the N:Pratio, size, and encapsulation efficiency should be struck.

In order to explore the efficacy of nanoparticle compositions withdifferent N:P ratios, the expression of luciferase (Luc) or humanerythropoietin (hEPO) in mice after low (0.05 mg/kg) or high (0.5 mg/kg)doses of intravenously administered nanoparticle compositions isexamined. The concentration of Luc or hEPO expressed is measured 3, 6,and/or 24 hours after administration.

Example 15: Optimization of Content of a Compound According to Formula(I), (IA), (II), (IIa), (IIb), (IIc), (IId) or (IIe)

As smaller particles with higher encapsulation efficiencies aregenerally desirable, the relative amounts of various elements in lipidcomponents of nanoparticle compositions are optimized according to theseparameters.

A compound according to Formula (I), (IA), (II), (IIa), (IIb), (IIc),(IId) or (IIe) is selected for optimization. The relative amount of thecompound according to Formula (I), (IA), (II), (IIa), (IIb), (IIc),(IId) or (IIe) is varied between 30 mol % and 60 mol % in compositionsincluding DOPE or DSPC as phospholipids to determine the optimal amountof the compound according to Formula (I), (IA), (II), (IIa), (IIb),(IIc), (IId) or (IIe) in the formulations. Formulations are preparedusing a standardized process with a water to ethanol ratio in thelipid-mRNA solution of 3:1 and a rate of injection of the lipid solutioninto the mRNA solution of 12 mL/min on a NanoAssemblr microfluidic basedsystem. This method induces nano-precipitation and particle formation.Alternative processes including, but not limited to, T-junction ordirect injection, may also be used to achieve the samenano-precipitation.

Formulations producing the smallest particles with the highestencapsulation efficiencies are generally preferred, however larger orsmaller particle sizes may be desirable based on a given application(e.g., based on the fenestration size of a target organ). Compositionsare also evaluated for their Luc or hEPO expression levels and cytokineprofiles.

Example 16: Optimization of Phospholipid

The relative amount of phospholipid in a lipid component of ananoparticle composition is varied to further optimize the formulation.A compound according to Formula (I), (IA), (II), (IIa), (IIb), (IIc),(IId) or (IIe) is selected for use in the nanoparticle composition andDOPE and DSPC are selected as phospholipids. Additional phospholipidscan also be evaluated. Nanoparticle compositions are prepared with therelative phospholipid content varying between 0 mol % and 30 mol %.Compositions are evaluated for their size, encapsulation efficiency, Lucor hEPO expression levels, and cytokine profiles.

Example 17: Optimization of Structural Lipid

The relative amount of structural lipid in a lipid component of ananoparticle composition is varied to further optimize the formulation.A compound according to Formula (I), (IA), (II), (IIa), (IIb), (IIc),(IId) or (IIe) is selected for use in the nanoparticle composition andcholesterol is selected as a structural lipid. Additional structurallipids can also be evaluated. Nanoparticle compositions are preparedwith the relative structural lipid content varying between 18.5 mol %and 48.5 mol %. Compositions are evaluated for their size, encapsulationefficiency, Luc or hEPO expression levels, and cytokine profiles.

Example 18: Optimization of PEG Lipid

The relative amount of PEG lipid in a lipid component of a nanoparticlecomposition is varied to further optimize the formulation. A compoundaccording to Formula (I), (IA), (II), (IIa), (IIb), (IIc), (IId) or(IIe) is selected for use in the nanoparticle composition and PEG-DMG isselected as a PEG lipid. Additional PEG lipids can also be evaluated.Nanoparticle compositions are prepared with the relative PEG lipidcontent varying between 0 mol % and 10 mol %. Compositions are evaluatedfor their size, encapsulation efficiency, Luc or hEPO expression levels,and cytokine profiles.

Exemplary formulations useful in the optimization of nanoparticlecomposition formulations are presented in Table 23.

TABLE 23 Exemplary formulations including compounds according to Formula(I), (IA), (II), (IIa), (IIb), (IIc), (IId) or (IIe). Composition (mol%) Components 40:20:38.5:1.5 Compound:Phospholipid:Chol:PEG-DMG45:15:38.5:1.5 Compound:Phospholipid:Chol:PEG-DMG 50:10:38.5:1.5Compound:Phospholipid:Chol:PEG-DMG 55:5:38.5:1.5Compound:Phospholipid:Chol:PEG-DMG 60:5:33.5:1.5Compound:Phospholipid:Chol:PEG-DMG 45:20:33.5:1.5Compound:Phospholipid:Chol:PEG-DMG 50:20:28.5:1.5Compound:Phospholipid:Chol:PEG-DMG 55:20:23.5:1.5Compound:Phospholipid:Chol:PEG-DMG 60:20:18.5:1.5Compound:Phospholipid:Chol:PEG-DMG 40:15:43.5:1.5Compound:Phospholipid:Chol:PEG-DMG 50:15:33.5:1.5Compound:Phospholipid:Chol:PEG-DMG 55:15:28.5:1.5Compound:Phospholipid:Chol:PEG-DMG 60:15:23.5:1.5Compound:Phospholipid:Chol:PEG-DMG 40:10:48.5:1.5Compound:Phospholipid:Chol:PEG-DMG 45:10:43.5:1.5Compound:Phospholipid:Chol:PEG-DMG 55:10:33.5:1.5Compound:Phospholipid:Chol:PEG-DMG 60:10:28.5:1.5Compound:Phospholipid:Chol:PEG-DMG 40:5:53.5:1.5Compound:Phospholipid:Chol:PEG-DMG 45:5:48.5:1.5Compound:Phospholipid:Chol:PEG-DMG 50:5:43.5:1.5Compound:Phospholipid:Chol:PEG-DMG 40:20:40:0Compound:Phospholipid:Chol:PEG-DMG 45:20:35:0Compound:Phospholipid:Chol:PEG-DMG 50:20:30:0Compound:Phospholipid:Chol:PEG-DMG 55:20:25:0Compound:Phospholipid:Chol:PEG-DMG 60:20:20:0Compound:Phospholipid:Chol:PEG-DMG 40:15:45:0Compound:Phospholipid:Chol:PEG-DMG 45:15:40:0Compound:Phospholipid:Chol:PEG-DMG 50:15:35:0Compound:Phospholipid:Chol:PEG-DMG 55:15:30:0Compound:Phospholipid:Chol:PEG-DMG 60:15:25:0Compound:Phospholipid:Chol:PEG-DMG 40:10:50:0Compound:Phospholipid:Chol:PEG-DMG 45:10:45:0Compound:Phospholipid:Chol:PEG-DMG 50:0:48.5:1.5Compound:Phospholipid:Chol:PEG-DMG 50:10:40:0Compound:Phospholipid:Chol:PEG-DMG 55:10:35:0Compound:Phospholipid:Chol:PEG-DMG 60:10:30:0Compound:Phospholipid:Chol:PEG-DMG

Example 19: Optimization of Particle Sizes

The fenestration sizes for different bodily organs often vary; forexample, the kidney is known to have a smaller fenestration size thanthe liver. Thus, targeting delivery of a therapeutic and/or prophylactic(e.g., specifically delivering) to a particular organ or group of organsmay require the administration of nanoparticle compositions withdifferent particle sizes. In order to investigate this effect,nanoparticle compositions with formulations such as those included inTable 23 are prepared with a variety of particle sizes using aNanoassemblr instrument. Nanoparticle compositions include an RNAencoding Luc. Each differently sized nanoparticle composition issubsequently administered to mice to evaluate the effect of particlesize on delivery selectivity. Luc expression in two or more organs orgroups of organs can be measured using bioluminescence to evaluate therelative expression in each organ.

Example 20: Administration Following Pretreatment

Administration of nanoparticle compositions to subjects can result ininflammation, infusion related reactions, and other undesirable effectsindicative of low tolerability. These effects can be attributed toundesirable immunoactivity.

In order to combat negative effects, nanoparticle compositions areco-administered with one or more substances (e.g., co-medications oradditional therapeutic and/or prophylactics) to subjects. Potentiallyuseful additional therapeutic and/or prophylactics include steroids(e.g., corticosteroids), anti-histamines, H1 receptor blockers, H2receptor blockers, anti-inflammatory compounds, statins, BTK inhibitors,S1P1 agonists, glucocorticoid receptor modulators (GRMs), andestradiols. Non-human primates are pretreated with one or moreadditional therapeutic agents selected from dexamethasone andacetaminophen. The additional therapeutic agent is administered either24 hours, 1 hour, or both 24 hours and 1 hour before administration of ananoparticle composition. Sample protocol are summarized in Table 24.Cytokine profiles, inflammation, and other parameters are measured andcompared to evaluate the effectiveness of pretreatment.

TABLE 24 Sample protocol for pretreatment study. Pretreatment AdditionalTherapeutic Agent(s) Group Time Administered 1 None None 2 24 hoursDexamethasone 3 24 hours Acetaminophen 4 24 hours Dexamethasone andAcetaminophen 5  1 hour Dexamethasone 6  1 hour Acetaminophen 7  1 hourDexamethasone and Acetaminophen 8 24 hours and 1 Dexamethasone hour 9 24hours and 1 Acetaminophen hour 10 24 hours and 1 Dexamethasone andAcetaminophen hour

For example, a useful therapeutic treatment course may involveadministering an additional therapeutic and/or prophylactic both the daybefore and the day of (one hour prior) to administration of ananoparticle composition at a dose level of 1.3 mpk. Additionaltherapeutic and/or prophylactics can be formulated for delivery by avariety of different routes. For example, dexamethasone may be deliveredorally. In general, additional therapeutic and/or prophylactics areadministered at clinically approved or typical dosage levels.

Example 21: Administration to Non-Human Primates

The tolerability and efficacy of nanoparticle compositions to non-humanprimates was evaluated in Cynomolgus monkeys. Monkeys were administeredan optimized nanoparticle composition including an mRNA encoding hEPOonce weekly for four weeks. The levels of hEPO protein, mRNA, andcytokine profiles were measured using ELISA-based techniques before and2, 6, 12, 24, 48, 72, and 120 hours after each administration.

The effects of pretreatment to non-human primates were evaluated using astandard MC3 formulation including an mRNA encoding hEPO. The studydesign is summarized in Table 25. Male monkeys were administered thenanoparticle composition once weekly for four weeks at a dose rate of 5ml/kg/h and were pretreated with either methotrexate or dexamethasone.

TABLE 25 Protocol for pretreatment study in Cynomolgus monkeys.Additional Dose Therapeutic Dose Test level Agent concentration Numberof Group Material (mg/kg) Administered (mg/ml) monkeys 1 MC3 0 None 0 32 hEPO 0.3 None 0.06 3 mRNA in MC3 3 hEPO 0.3 Methotrexate 0.06 3 mRNAin MC3 4 hEPO 0.3 Dexamethasone 0.06 3 mRNA in MC3

Results of the pretreatment study are shown in FIG. 1. As shown, in theabsence of any pretreatment, maximal expression levels decreased nearly70% over the course of the study. Methotrexate did not confer anyparticular beneficial effect. However, pre-administration ofdexamethasone resulted in increased protein expression compared totreatment courses not involving pretreatment. Notably, minimal decreasein plasma/serum protein expression was observed over time for animalspretreated with dexamethasone. These results suggest that pretreatmentof corticosteroids such as dexamethasone improves the tolerability andefficacy of nanoparticle compositions containing, for example, acompound according to Formula (I), (IA), (II), (IIa), (Ib), (IIc), (IId)or (IIe).

The tolerability and efficacy of nanoparticle compositions to non-humanprimates was also investigated using a sample formulation includingCompound 18. The formulation was prepared according to the standard MC3formulation described above and included an hEPO mRNA. Primates wereadministered a single dose of 0.05 (Group 1), 0.3 (Group 2), or 1.0(Group 3) mpk via intravenous infusion for 60 minutes. Three primateswere administered each dose. Expression of hEPO was measured prior todosing and at 2, 6, 24, 48, and 96 hours post-treatment (Table 26).Pharmacokinetic parameters including Tmax, Cmax, and the AUC were alsodetermined and are presented in Table 27. Table 28 includes levels ofindicators of complement activation, while Table 29 includes cytokineinduction data.

TABLE 26 hEPO expression measured at various time points uponadministration of nanoparticle compositions to non-human primates. hEPOconcentration Group 1 Group 2 Group 3 (pg/ml) (0.05 mpk) (0.3 mpk) (1.0mpk) Predose 1000 1000 1000  2 h 142588 363272 312006  6 h 379362 341285502663 24 h 103055 148789 467598 48 h 25382 57095 175953 96 h 2084 609524795

TABLE 27 Pharmacokinetic parameters measured upon administration ofnanoparticle compositions to non-human primates. Group 1 Group 2 Group 3(0.05 mpk) (0.3 mpk) (1.0 mpk) Tmax Mean 6.00 3.33 12.0 (hours) SD 0.002.31 10.4 CV % 0.00 69.3 86.6 Cmax Mean 3.79E+05 3.84E+05 5.51E+05(pg/ml) SD 2.64E+05 2.45E+05 6.24E+04 CV % 69.7 63.8 11.3 AUCall Mean7.72E+06 1.02E+07 2.32E+07 (hr · pg/ml) SD 6.26E+06 7.34E+06 4.20E+06 CV% 81.1 72.3 18.1

TABLE 28 Complement activation indicators measured at various timepoints upon administration of nanoparticle compositions to non-humanprimates. Group 1 Group 2 Group 3 Time point (0.05 mpk) (0.3 mpk) (1.0mpk) C3a Predose 10600 9827 12792 (ng/ml) 2 h 19236 42897 75936 6 h12385 32436 51996 24 h  11596 19721 35843 Day 5 11945 16207 19101 BbPredose 1375 1461 1529 fragment 2 h 5341 5356 8849 (ng/ml) 6 h 3037 715712820 24 h  1496 3680 8601 Day 5 1273 2400 2834 C5b9 Predose 169 157 238(ng/ml) 2 h 1959 393 801 6 h 786 1333 2928 24 h  265 614 4798 Day 5 163405 534

TABLE 29 Cytokine induction measured at various time points uponadministration of nanoparticle compositions to non-human primates. Group1 Group 2 Group 3 Time point (0.05 mpk) (0.3 mpk) (1.0 mpk) IFNg Predose18.8 18.8 18.8 (pg/ml) 2 h 18.8 18.8 35.8 6 h 18.8 18.8 38.9 24 h  18.818.8 18.8 Day 5 39.3 18.8 18.8 IFNα Predose 18.8 18.8 18.8 (pg/ml) 2 h18.8 18.8 18.8 6 h 18.8 18.8 18.8 24 h  18.8 18.8 18.8 Day 5 18.8 18.818.8 IL-1b Predose 18.8 18.8 18.8 (pg/ml) 2 h 18.8 18.8 33.4 6 h 18.818.8 18.8 24 h  18.8 18.8 18.8 Day 5 18.8 18.8 18.8 IL-6 Predose 18.818.8 18.8 (pg/ml) 2 h 18.8 191 834 6 h 18.8 33.0 398 24 h  18.8 18.831.4 Day 5 18.8 18.8 18.8 MCP-1 Predose 192 168 235 (pg/ml) 2 h 342 30184221 6 h 543 2011 3945 24 h  236 404 1444 Day 5 232 211 225 TNF-αPredose 18.8 18.8 18.8 (pg/ml) 2 h 18.8 38.2 18.8 6 h 41.5 32.5 18.8 24h  17.6 59.6 46.2 Day 5 63.5 18.8 41.9

In general, the formulation was tolerated similarly to the MC3formulation with dose-response effects. Aspartate aminotransferase (AST)increased in the high dose group on Day 2 and returned to baseline byDay 5. Alanine aminotransferase (ALT) levels did not increase, however.In general, lower doses were better tolerated. High doses induced bodytemperature elevation and hunched posture, which is similar to thebehavior observed for primates administered higher doses of MC3formulations. White blood cell counts were slightly elevated in animalsin the high dose group, however all groups showed a marked increase inreticulocyte counts on Day 5, indicating a strong pharmacologicalresponse. Complement activation and cytokine release (IL-6 and MCP-1)were dose-related and reversible within 24 hours for the low andmid-dose groups and by Day 5 in the high dose group. hEPO levels werehigher than those measured upon administration of comparable doses ofMC3 formulations to non-human primates.

Example 22: Administration to Non-Human Primates

The tolerability and efficacy of nanoparticle compositions to non-humanprimates was also investigated using sample formulations includingCompounds 18, 25, 26, and 48 and MC3 to determine if these compounds aredifferentiated in terms of protein expression. The formulations wereprepared according to the standard MC3 formulation described above andincluded an hEPO mRNA. Table 30 includes details of the compositionstested, while Table 31 summarizes the relative expression of Luc andhEPO mRNA in mice and rats. Expression was measured 6 hours afteradministration.

TABLE 30 Characteristics of nanoparticle compositions includingcompounds according to Formula (I), (IA), (II), (IIa), (IIb), (IIc),(IId) or (IIe). Zeta Size EE Osmolality potential Compound (nm) PDI (%)pH (mOsm/kg) (mV) 18 102.6 0.230 85.56 7.64 312 −3.53 25 98.8 0.23087.01 7.60 304 −3.88 26 79.2 0.120 95.60 7.54 305 −3.73 48 70.6 0.17691.92 7.58 311 −3.61 MC3 106.0 0.220 91.66 7.52 318 −3.64 n.d. = notdetermined

TABLE 31 Comparison of nanoparticle compositions including compoundsaccording to Formula (I), (IA), (II), (IIa), (IIb), (IIc), (IId) or(IIe). Compound Compound Compound Compound 18 25 26 48 MC3 Lipid/MC3 Lucexpression 3.23 1.89 4.24 10.0 1 (0.5 mpk dose to mouse) Lipid/MC3 hEPOn.d. 2.41 n.d. 5.39 1 expression (0.2 mpk dose to rat) Lipid/MC3 hEPO0.81 2.13 4.95 3.84 1 expression (2 mpk dose to rat) % lipid remainingin liver 0.018 1.32 20 11.4 87 after 48 hours n.d. = not determined

FIG. 2 shows the hEPO mRNA expression measured after intravenousadministration of a 0.01 mpk dose with 60 minutes infusion to naivecynomolgus monkeys. As is evident in the figure, expression was highest6 hours post administration for all formulations tested, and was highestfor those formulations including Compound 18.

Table 32 summarizes pharmacokinetic parameters measured uponadministration of 0.01 mpk doses of formulations to non-human primates.

TABLE 32 Pharmacokinetic parameters measured upon administration offormulations including compounds according to Formula (I), (IA), (II),(IIa), (IIb), (IIc), (IId) or (IIe) to non-human primates. AUC T_(max)(hr) C_(max) (ng/mL) AUC_(0-t) (hr*ng/mL) Lipid/MC3 Lipid Mean Mean SDCV % Mean SD CV % Ratio MC3 8 14.1 2.36 16.8 284 97.4 34.2 1.0 Compound18 8 91 34.7 38.1 1690 1060 63.1 6.0 Compound 25 6 56.9 15.1 26.5 930249 26.8 3.3 Compound 26 6 28.2 17.7 62.7 365 302 82.7 1.3 Compound 48 617.7 9.49 53.6 245 117 47.9 0.9

Example 23: Administration to Non-Human Primates

Results of hEPO expression studies were validated using a standard MC3formulation and a nanoparticle composition containing Compound 18including an mRNA encoding an anti-hemagglutinin (anti-HA) antibody.Cynomolgus monkeys were administered a single dose of 0.1 mpk or 0.3 mpkof a nanoparticle composition containing Compound 18 (see Table 33;prepared according to Example 2) including an mRNA encoding anti-HAantibody via intravenous infusion for 60 minutes.

TABLE 33 Diameter Lipid EE (%) (nm) PDI Compound 18 79.3 76.8 0.16

The results of anti-HA (anti-hemagglutinin) antibody expression areshown in Table 34 and in FIG. 11.

TABLE 34 Dose AUC AUC Compound Lipid (mpk) (μg/mL * h) 18/MC3 Ratio MC30.1 77.05 — Compound 18 0.1 354.3 4.6 MC3 0.3 235.7 — Compound 18 0.31055 4.5

A five times higher protein expression was observed with thenanoparticle composition containing Compound 18 versus the MC3counterpart, and a clear dose response between 0.1 and 0.3 mpk withCompound 18 was found (e.g., 0.3 mpk AUC is about three times of thatfrom 0.1 mpk dose).

Example 24: Methods of Treating Diseases and Disorders

A nanoparticle composition formulation having high tolerability (e.g.,provoking a low immune response) and efficacy (e.g., facilitatingefficient and effective encapsulation of a therapeutic and/orprophylactic and delivery of the agent to a desired target) is selectedfor use. A therapeutic and/or prophylactic for formulation with thenanoparticle composition is selected for use based on the condition of asubject. For example, an mRNA encoding a vascular endothelial growthfactor A (VEGF-A) may be selected to promote angiogenesis to treatatherosclerotic renovascular disease, while an siRNA capable of knockingdown apolipoprotein B (apoB) may be selected to treat a metabolicdisease or disorder such as dyslipidemia.

A subject in need of treatment is pretreated with a small dose ofdexamethasone one or more hours prior to treatment with the nanoparticlecomposition. The nanoparticle composition is preferably administered tothe subject intravenously, however intramuscular, intradermal,subcutaneous, intranasal, or inhalation administration routes are alsoacceptable. Treatment is provided in a dose of about 0.001 mg/kg toabout 10 mg/kg of therapeutic and/or prophylactic and is repeated daily,weekly, biweekly, or monthly according to needs of the subject.

EQUIVALENTS

It is to be understood that while the present disclosure has beendescribed in conjunction with the detailed description thereof, theforegoing description is intended to illustrate and not limit the scopeof the present disclosure, which is defined by the scope of the appendedclaims. Other aspects, advantages, and alterations are within the scopeof the following claims.

1. (canceled)
 2. A compound of Formula (IA):

or a salt or isomer thereof, wherein l is selected from 1, 2, 3, 4, and5; m is selected from 5, 6, 7, 8, and 9; M₁ is a bond or M′; R₄ isunsubstituted C₁₋₃ alkyl, or —(CH₂)_(n)Q, in which Q is OH,—NHC(S)N(R)₂, —NHC(O)N(R)₂, —N(R)C(O)R, —N(R)S(O)₂R, —N(R)R₈,—NHC(═NR₉)N(R)₂, —NHC(═CHR₉)N(R)₂, —OC(O)N(R)₂, —N(R)C(O)OR,—N(OR)C(O)R, —N(OR)S(O)₂R, —N(OR)C(O)OR, —N(OR)C(O)N(R)₂,—N(OR)C(S)N(R)₂, —N(OR)C(═NR₉)N(R)₂, —N(OR)C(═CHR₉)N(R)₂, or heteroaryl,and each n is selected from 1, 2, 3, 4, or 5; M and M′ are independentlyselected from —C(O)O—, —OC(O)—, —C(O)N(R′)—, —P(O)(OR′)O—, —S—S—, anaryl group, and a heteroaryl group; and R₂ and R₃ are both C₁₋₁₄ alkyl,or C₂₋₁₄ alkenyl, R₈ is selected from the group consisting of C₃₋₆carbocycle and heterocycle; R₉ is selected from the group consisting ofH, CN, NO₂, C₁₋₆ alkyl, —OR, —S(O)₂R, —S(O)₂N(R)₂, C₂₋₆ alkenyl, C₃₋₆carbocycle and heterocycle; each R is independently selected from thegroup consisting of C₁₋₃ alkyl, C₂₋₃ alkenyl, and H; and R′ is a linearalkyl.
 3. (canceled)
 4. The compound of claim 2, wherein the compound isof Formula (II):

or a salt or isomer thereof, wherein n is 2, 3, or
 4. 5.-14. (canceled)15. The compound of claim 2, wherein R₄ is unsubstituted methyl or—(CH₂)_(n)Q, in which Q is OH, —NHC(S)N(CH₃)₂, —NHC(S)NH(CH₃),—NHC(O)NH(CH₃), or —NHC(O)N(CH₃)₂. 16.-44. (canceled)
 45. The compoundof claim 2, wherein R₂ and R₃ are the same.
 46. The compound of claim 2,wherein R₂ and R₃ are C₈ alkyl. 47.-52. (canceled)
 53. The compoundclaim 2, wherein R₂ and R₃ are different. 54.-111. (canceled)
 112. Thecompound of claim 2, wherein the compound is selected from the groupconsisting of Compounds 1-5, 7, 8, 19-20, 23, 28, 30, 32-47, 56-61, 96,100, 104, 107-117, 122-124, 126-129, 131, 133-134, 143-147, 153,168-170, 173-179, 181-182, 185, 186, 193, 194, 196-198, 200, 203-213,215, 218-228, 230, and 231, and salts and isomers thereof. 113-115.(canceled)
 116. The compound of claim 2, wherein the compound is of theFormula (IIa),

or a salt or isomer thereof. 117.-121. (canceled)
 122. The compound ofclaim 2, wherein the compound is of the Formula (IId),

or a salt or isomer thereof, wherein n is selected from 2, 3, and 4.123.-131. (canceled)
 132. A nanoparticle composition comprising a lipidcomponent comprising a compound of claim
 2. 133. The nanoparticlecomposition of claim 132, wherein the lipid component further comprisesa phospholipid selected from the group consisting of1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC),1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC),1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC),1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC),1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC),1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC),1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC),1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine(OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C₁₆ Lyso PC),1,2-dilinolenoyl-sn-glycero-3-phosphocholine,1,2-diarachidonoyl-sn-glycero-3-phosphocholine,1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine,1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE),1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE),1,2-distearoyl-sn-glycero-3-phosphoethanolamine,1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine,1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine,1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine,1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine,1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG),sphingomyelin, and mixtures thereof. 134.-136. (canceled)
 137. Thenanoparticle composition of claim 132, wherein the lipid componentfurther comprises a structural lipid selected from the group consistingof cholesterol, fecosterol, sitosterol, ergosterol, campesterol,stigmasterol, brassicasterol, tomatidine, ursolic acid,alpha-tocopherol, and mixtures thereof. 138.-139. (canceled)
 140. Thenanoparticle composition of claim 132, wherein the lipid componentfurther comprises a PEG lipid selected from the group consisting of aPEG-modified phosphatidylethanolamine, a PEG-modified phosphatidic acid,a PEG-modified ceramide, a PEG-modified dialkylamine, a PEG-modifieddiacylglycerol, a PEG-modified dialkylglycerol, and mixtures thereof.141. (canceled)
 142. The nanoparticle composition of claim 132, whereinthe lipid component further comprises a cationic and/or ionizable lipidselected from the group consisting of3-(didodecylamino)-N1,N1,4-tridodecyl-1-piperazineethanamine (KL10),N1-[2-(didodecylamino)ethyl]-N1,N4,N4-tridodecyl-1,4-piperazinediethanamine (KL22),14,25-ditridecyl-15,18,21,24-tetraaza-octatriacontane (KL25),1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA),2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA),heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate(DLin-MC3-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane(DLin-KC₂-DMA), 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA),2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (Octyl-CLinDMA),(2R)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine(Octyl-CLinDMA (2R)), and(2S)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine(Octyl-CLinDMA (2S)).
 143. (canceled)
 144. The nanoparticle compositionof claim 132, wherein the lipid component comprises about 30 mol % toabout 60 mol % said compound, about 0 mol % to about 30 mol %phospholipid, about 18.5 mol % to about 48.5 mol % structural lipid, andabout 0 mol % to about 10 mol % PEG lipid.
 145. (canceled)
 146. Thenanoparticle composition of claim 132, further comprising a therapeuticand/or prophylactic agent. 147.-148. (canceled)
 149. The nanoparticlecomposition of claim 132, wherein the therapeutic and/or prophylacticagent is a ribonucleic acid (RNA), wherein the RNA is selected from thegroup consisting of a small interfering RNA (siRNA), an asymmetricalinterfering RNA (aiRNA), a microRNA (miRNA), a Dicer-substrate RNA(dsRNA), a small hairpin RNA (shRNA), a messenger RNA (mRNA), andmixtures thereof.
 150. (canceled)
 151. The nanoparticle composition ofclaim 132, wherein the RNA is an mRNA.
 152. The nanoparticle compositionof claim 132, wherein the mRNA includes one or more of a stem loop, achain terminating nucleoside, a polyA sequence, a polyadenylationsignal, and/or a 5′ cap structure.
 153. (canceled)
 154. The nanoparticlecomposition of claim 132, wherein the encapsulation efficiency of thetherapeutic and/or prophylactic agent is at least 80% or at least 90%.155. (canceled)
 156. The nanoparticle composition of claim 132, whereinthe wt/wt ratio of the lipid component to the therapeutic and/orprophylactic agent is from about 10:1 to about 60:1.
 157. (canceled)158. The nanoparticle composition of claim 132, wherein the N:P ratio isfrom about 2:1 to about 30:1. 159.-162. (canceled)
 163. A pharmaceuticalcomposition comprising the nanoparticle composition of claim 132 and apharmaceutically acceptable carrier.
 164. A method of delivering atherapeutic and/or prophylactic agent to a mammalian cell, the methodcomprising administering to a subject the nanoparticle composition ofclaim 132, said administering comprising contacting the cell with thenanoparticle composition, whereby the therapeutic and/or prophylacticagent is delivered to the cell. 165.-188. (canceled)